Glp-1, exendin-4, peptide analogs and uses thereof

ABSTRACT

The invention relates to novel polypeptide analogs of GLP-1 and exendin-4. The polypeptide, in a preferred embodiment, is insulinotropic and long-acting. Preferably, the polypeptide&#39;s insulinotropic effect is comparable to or exceeds the effect of an equimolar amount of GLP-1 or exendin-4. The invention also relates to a method of treating a subject with diabetes, comprising administering to the subject the polypeptide of the invention in an amount that has an insulinotropic effect. The invention also relates to methods of using GLP-1, exendin-4, and polypeptide analogs thereof for neuroprotective and neurotrophic effects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.13/594,313, filed Aug. 24, 2012, which is a continuation application ofU.S. Ser. No. 12/317,042, filed Dec. 18, 2008 (now U.S. Pat. No.8,278,272), which is a continuation application of U.S. Ser. No.10/485,140, filed Apr. 1, 2004 (now U.S. Pat. No. 7,576,050), which is anational stage application filed under 35 U.S.C. §371 of internationalapplication no. PCT/US2002/024141, filed Jul. 30, 2002, which claims thebenefit of priority under 35 U.S.C. §119(e) to U.S. ProvisionalApplication Ser. No. 60/309,076, filed Jul. 31, 2001, each of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to glucagon-like peptide-1 (GLP-1),exendin-4 and their peptide analogs. The invention also relates to theiruses in the treatment of diabetes and neurodegenerative conditions.

2. Background Art

Pancreatic beta cell dysfunction and the concomitant decrease in insulinproduction can result in diabetes mellitus. In type 1 diabetes, the betacells are completely destroyed by the immune system, resulting in anabsence of insulin producing cells (Physician's Guide to InsulinDependent [Type I] Diabetes Mellitus: Diagnosis and Treatment, AmericanDiabetes Association, 1988). In type 2 diabetes, the beta cells becomeprogressively less efficient as the target tissues become resistant tothe effects of insulin on glucose uptake. Thus, beta cells are absent inpeople with type 1 diabetes and are functionally impaired in people withtype 2 diabetes.

Beta cell dysfunction currently is treated in several different ways. Inthe treatment of type 1 diabetes or the late stages of type 2 diabetes,insulin replacement therapy is necessary. Insulin therapy, althoughlife-saving, does not restore normoglycemia, even when continuousinfusions or multiple injections are used in complex regimes. Forexample, postprandial levels of glucose continue to be excessively highin individuals on insulin replacement therapy. Thus, insulin therapymust be delivered by multiple daily injections or continuous infusionand the effects must be carefully monitored to avoid hyperglycemia,hypoglycemia, metabolic acidosis, and ketosis.

People with type 2 diabetes are generally treated with drugs thatstimulate insulin production and secretion from the beta cells and/orimprove insulin sensitivity. A major disadvantage of these drugs,however, is that insulin production and secretion is promoted regardlessof the level of blood glucose. Thus, food intake must be balancedagainst the promotion of insulin production and secretion to avoidhypoglycemia or hyperglycemia. In recent years several new agents havebecome available to treat type 2 diabetes. These include metformin,rosiglitazone, pioglitazone, and acarbose (see Bressler and Johnson,1997). However, the drop in hemoglobin A1c obtained by these neweragents is less than adequate (Ghazzi et al., 1997), suggesting that theywill not improve the long-term control of diabetes mellitus.

Glucagon-like peptide-1 (GLP-1), a hormone normally secreted byneuroendocrine cells of the gut in response to food, has been suggestedas a new treatment for type 2 diabetes (Gutniak et al., 1992; Nauck etal., J. Clin. Invest., 1993). It increases insulin release by the betacells even in subjects with long-standing type 2 diabetes (Nauck et al.,Diabetologia, 1993). GLP-1 treatment has an advantage over insulintherapy because GLP-1 stimulates endogenous insulin secretion, whichturns off when blood glucose levels drop (Nauck et al., Diabetologia,1993; Elahi et al., 1994). GLP-1 promotes euglycemia by increasinginsulin release and synthesis, inhibiting glucagon release, anddecreasing gastric emptying (Nauck et al., Diabetologia, 1993; Elahi etal., 1994; Wills et al., 1996; Nathan et al., 1992; De Ore et al.,1997). GLP-1 also induces an increase in hexokinase messenger RNA levels(Wang et al., Endocrinology 1995; Wang et al., 1996). GLP-1 is known tohave a potent insulin-secreting effect on beta cells (Thorens andWaeber, 1993; Orskov, 1992) and to increase insulin biosynthesis andproinsulin gene expression when added to insulin-secreting cell linesfor 24 hours (Drucker et al., 1987; Fehmann and Habener, 1992). Instudies using RIN 1046-38 cells, twenty-four hour treatment with GLP-1increased glucose responsiveness even after the GLP-1 had been removedfor an hour and after several washings of the cells (Montrose-Rafizadehet al., 1994). Thus, GLP-1 is an insulinotropic agent known to havebiological effects on βcells even after it has been metabolized from thesystem. GLP-1 is a product of posttranslational modification ofproglucagon. The sequences of GLP-1 and its active fragments GLP-1(7-37) and GLP-1(7-36) amide are known in the art (Fehmann et al.,1995). Although GLP-1 has been proposed as a therapeutic agent in thetreatment of diabetes, it has a short biological half-life (De Ore etal., 1997), even when given by a bolus subcutaneously (Ritzel et al.,1995). GLP-1 degradation (and GLP-1 (7-36) amide), in part, is due tothe enzyme dipeptidyl peptidase (DPP1V), which cleaves the polypeptidebetween amino acids 8 and 9 (alanine and glutamic acid).

Exendin-4 is a polypeptide produced in the salivary glands of the GilaMonster lizard (Goke et al., 1993). The amino acid sequence forexendin-4 is known in the art (Fehmann et al. 1995). Although it is theproduct of a uniquely non-mammalian gene and appears to be expressedonly in the salivary gland (Chen and Drucker, 1997), exendin-4 shares a52% amino acid sequence homology with GLP-1 and in mammals interactswith the GLP-1 receptor (Goke et al., 1993; Thorens et al., 1993). Invitro, exendin-4 has been shown to promote insulin secretion by insulinproducing cells and, given in equimolar quantities, is more potent thanGLP-1 at causing insulin release from insulin producing cells.Furthermore, exendin-4 potently stimulates insulin release to reduceplasma glucose levels in both rodents and humans and is longer actingthan GLP-1. Exendin-4, however, because it does not occur naturally inmammalians, has certain potential antigenic properties in mammals thatGLP-1 lacks.

In addition to the reduction in insulin production that occurs indiabetes, peripheral neuropathy is commonly associated with diabetes.Twenty to thirty percent of all diabetes subjects eventually developperipheral neuropathy. Furthermore, there are reports of increased riskof Alzheimer's disease with heart disease, stroke, hypertension, anddiabetes (Moceri et al., 2000; Ott et al., 1999). Thus, diabetes is adisease that is also associated with neurodegenerative diseases.

A number of studies have demonstrated that the GLP-1 receptor is presentin both the rodent (Jin et al 1988, Shughrue et al 1996) and human (Weiand Mojsov 1995, Satoh et al 2000) brains. The chemoarchitecture of thedistribution appears to be largely confined to the hypothalamus,thalamus, brainstem, lateral septum, the subfomical organ and the areapostrema, all circumventricular areas where generally large numbers ofpeptide receptors are located. However, specific binding sites for GLP-1have also been detected throughout the caudate-putamen, cerebral cortexand cerebellum (Campos et al. 1994, Calvo et al. 1995, Goke et al.1995), albeit at low densities.

Needed in the art are polypeptides that are of therapeutic value in thetreatment of diabetes and the treatment of degenerative disorders suchas Alzheimer's and Parkinson's diseases, as well as the peripheralneuropathy associated with type 2 diabetes mellitus.

SUMMARY OF THE INVENTION

In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates tonovel polypeptide analogues of GLP-1 and exendin-4. The polypeptide, ina preferred embodiment, is insulinotropic and long-acting. Preferably,the polypeptide's insulinotropic effect is comparable to or exceeds theeffect of an equimolar amount of GLP-1 or exendin-4.

The invention further relates to a purified polypeptide, the amino acidsequence of which comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:25, or SEQ ID NO:33.

In another aspect, the invention relates to a method of treating asubject with diabetes, comprising administering to the subject thepolypeptide of the invention in an amount that has an insulinotropiceffect.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate (one) several embodiment(s) ofthe invention and together with the description, serve to explain theprinciples of the invention.

FIG. 1A, FIG. 1B, and FIG. 1C are schematics showing sequences for the35 synthetic polypeptides tested for their insulinotropic properties andthe sequences for GLP-1 and Ex-4. Dark shading shows exendin-4-likeresidues and light shading shows GLP-1-like residues.

FIG. 2 shows a comparison in insulin secretion in RIN 1048-36 cells inthe presence of GLP-1, exendin-4, and the synthetic polypeptidesidentified in FIG. 1. Levels are expressed as a percentage of basallevels.

FIG. 3 shows a comparison in insulin secretion in RIN 1048-38 cells inthe presence of glucose (5 mM) and in the presence or absence of 10 nMGLP-1 (SEQ ID NO:1, GLP-1 Gly⁸ (peptide 1; SEQ ID NO:3), GLP-16-aminohexanoic acid⁸ (peptide 11; SEQ ID NO:8), GLP-1 (6-aminohexanoicacid⁹)₄ (peptide 25; SEQ ID NO:22), GLP-1 (6-aminohexanoic acid⁹)₈(peptide 26; SEQ ED NO: 23), or six analogues of GLP-1 that contain,from the carboxy terminus, 3, 5, 7, 12, 21, and all D-amino acids. Thedata represent the mean of 2-3 experiments ±SEM. **p<0.001, *p<0.05 fortreated versus basal. Levels are expressed in pg of insulin/μg ofprotein. Basal release is also shown.

FIG. 4 shows the effect of GLP-1 analogs on the production ofintracellular cAMP. CHO/GLP-1R cells were incubated with the indicatedpolypeptides (10 nM) for 30 min at 37° C., after which they were lysedand the lysates processed for determination of cAMP content. The dataare normalized to maximum values obtained in the presence of GLP-1 (10nM). The data points represent the mean of 2-3 experiments. **p<0.001,*p<0.05 for treated versus basal.

FIG. 5 shows dose response curves for GLP-1, GLP-1 Gly⁸ (SEQ ID NO:3),and GLP-1 6-aminohexanoic acid⁸ (SEQ ID NO:8). Intracellular cAMP levelswere measured in CHO/GLP-1R cells after treatment with the indicatedconcentrations of GLP-1, GLP-1 Gly⁸, and GLP-1 6-aminohexanoic acid⁸ for30 min at 37° C. The data were normalized to maximum values obtained ineach experiment in the presence of GLP-1 (10 nM). Bars represent themeans±SEM of three experiments preformed in triplicate.

FIG. 6A, FIG. 6B, and FIG. 6C are dot plots showing the displacement of[¹²⁵I]-GLP-1 binding to CHO/GLP-1R cells with analogs of GLP-1.[¹²⁵I]-GLP-1 binding to intact CHO/GLPR cells was competed with variousconcentrations of the polypeptides shown. The data are normalized tomaximum values obtained in the presence of 10 nM of the respectivepolypeptides. The data points represent the mean±SEM of threeexperiments preformed in triplicate.

FIG. 7 shows the acute insulin-secreting activity of 0.4 nmol/kg ofpolypeptide Ex-4 WOT (SEQ ID NO:7) and GLP-1 Gly⁸ (SEQ ID NO:3) infasted, diabetic Zucker rats to induce insulin secretion as compared toequimolar concentrations of exendin-4 and GLP-1.

FIG. 8 shows the time course of insulin-secreting activity of 0.4nmol/kg of polypeptide 10 (Ex-4 WOT (SEQ ID NO:7)), polypeptide 1 (GLP-1Gly⁸ (SEQ ID NO:3)), and polypeptide 11 (GLP-1 6-aminohexanoic acid⁸(SEQ ID NO:8)) in fasted, diabetic Zucker rats up to 24 hours ascompared to equimolar concentrations of exendin-4 and GLP-1.

FIG. 9A and FIG. 9B are line graphs showing the biological effects ofGLP-1 Gly⁸ (SEQ ID NO:3) and GLP-1 6-aminohexanoic acid⁸ (SEQ ID NO:8).FIG. 9A shows the effect on blood glucose levels and FIG. 9B shows theeffect on insulin levels following subcutaneous injection of GLP-16-aminohexanoic acid⁸ (24 nmol/kg) to Wistar and Zucker fatty rats andGLP-1 Gly⁸ (24 nmol/kg) to Zucker rats only. Both Zucker and Wistar ratswere fasted overnight prior to injection. The results are means±SEM, n=6per group.

FIG. 10A, FIG. 10B, and FIG. 10C are a series of dot plots showing thedisplacement of [¹²⁵I] GLP-1 binding to CHO/GLP-1R cells with theanalogs of GLP-1, GLP-1 Gly8 and Ex-4. [¹²⁵I] GLP-1 binding to intactCHO/GLP-1R cells was competed with various concentrations of thepeptides shown. Each of FIG. 10A, FIG. 10B, and FIG. 10C show the datafor different peptides. The data are normalized to maximum valuesobtained in the presence of 10 nM of the respective peptides. The datapoints respresent the mean of three experiments performed in triplicate.B_(o), maximum binding in the absence of cold peptide.

FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D are a series of bar chartsshowing the densitometric quantification of proteins extracted from NGF,exendin-4, exendin-4 WOT and GLP-1 treated PC12 cells. Protein bandsobtained from cell lysates and conditioned media samples were analyzedby Western blotting and immunoprobed with the 22C11 monoclonal antibody(epitope: βAPP aa 66-81, Roche Molecular Biochemicals, Indianapolis,Ind.). Data are presented as the percent change in expression of βAPPderivatives from cell lysates samples (FIG. 11A and FIG. 11B) andsoluble sAPP from conditioned media samples taken on day 3 of treatment(FIG. 11C and FIG. 11D) relative to untreated control samples culturedin low serum media alone. Vertical error bars represent standard errorof 3 individual experimental values. Significant difference fromuntreated: *p<0.05 and **p<0.01.

FIG. 12 shows the effect of different concentrations of NGF and/orexendin-4 treatment on neurite outgrowth in PC12 cells. Neuriteoutgrowth is represented as the percent increase in number of cellsbearing neurites relative to untreated (low serum medium). Verticalerror bars represent ±standard error of the difference between the meansof six individual experimental values. Significant difference fromuntreated: *p<0.05 and **p<0.01.

FIG. 13 shows the effect of exendin-4 treatment on NGF-mediated celldeath. Combination treatments were carried out for a total of 7 days, inthe presence or absence of 50 ng/ml NGF, with or without exendin-4 (at 1or 5 mg/ml). Cells were subsequently harvested and allowed to rejuvenatein complete media for an additional 3 days. Cell survival is presentedas the proportion of viable cells (by MTT method) on day 10. Verticalerror bars represent ±standard error of four individual experimentalvalues.

FIG. 14A and FIG. 14B are bar graphs showing densitometricquantification of the synaptophysin protein extracted from NGF,exendin-4, exendin-4 WOT and GLP-1 treated PC12 cells. Protein bandsobtained from cell lysate samples were analyzed by Western blotting andimmunoprobed with the synaptophysin monoclonal antibody, which stainsneurosecretory vesicles. Synaptophysin was used as a marker ofdifferentiation. Density of the synaptophysin protein is presented asthe percent difference from untreated. Vertical error bars represent±standard error of three individual experimental values conducted atseparate time intervals. Significant difference from untreated:**p<0.01.

FIG. 15A and FIG. 15B are a series of bar charts showing fold increasesin lactate dehydrogenase (LDH) levels in the conditioned medium of PC12cells following treatment with NGF, exendin-4, exendin-4 WOT and GLP-1.LDH levels are a marker of cell viability, with elevated levels beingassociated with a loss of cell integrity. Vertical error bars represent±standard error of the difference between the means of three individualexperimental values conducted at separate time intervals. Significantdifference from untreated: *p<0.05 and **p<0.01.

FIG. 16A, FIG. 16B, and FIG. 16C are a series of line graphs and a barchart showing displacement of ¹²⁵I-GLP-1 binding with cold GLP-1 (FIG.16A), GLP-1 stimulated release of cAMP (FIG. 16B) and protection againstglutamate-induced apoptosis (FIG. 16C) in cultured hippocampal neurons.¹²⁵I-GLP-1 binding to intact cultured hippocampal neurons was competedwith various concentrations of GLP-1. The data are normalized to maximumvalues obtained in the presence of 1 μM GLP-1. Each data pointrepresents the mean of two experimental values and is presented as thepercentage of maximum binding in the absence of cold peptide. cAMPlevels were assayed over 30 min incubation with 10 nM GLP-1 (FIG. 16B).Vertical error bars represent ±standard error of the mean of threeindividual experimental values. Treatment with 10 nM GLP-1 or 0.3 μMexendin-4 completely protected against the apoptotic effects of 10 μMglutamate (FIG. 16C). Cultures were treated overnight, fixed with 4%paraformaldehyde and stained with Hoechst 33342. The number of apoptoticnuclei were counted and the values are presented as the pooled mean ofsix individual dishes per treatment condition. Vertical error barsrepresent ±standard error of the difference between the means.Significant difference from control: *p<0.05, **p<0.01 and ***p<0.001.

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F are aseries of photomicrographs showing choline acetyltransferase and glialfibrillary acidic protein immunoreactivity in the basal nucleus.ChAT-positive immunoreactivity, ipsilateral (FIG. 17A and FIG. 17C) andcontralateral (FIG. 17B and FIG. 17D), to a partial ibotenic acidlesion. FIG. 17A & FIG. 17B and FIG. 17C & FIG. 17D depict the left andright basal nucleus from individual animals which received vehicleinfusion and GLP-1 infusion, respectively. ChAT-positiveimmunoreactivity in the ipsilateral basal nucleus in an animal whichreceived vehicle infusion (FIG. 17A) was substantially lower than thatin the ipsilateral basal nucleus in an animal which received GLP-1infusion (FIG. 17C). Glial fibrillary acidic protein (GFAP)immunoreactivity, a marker for reactive astrocytes produced in responseto injury, demonstrated areas of positive immunoreactivity surroundingthe site of cannula implantation and lining of the lateral ventricle inthe vicinity of the site of infusion. Interestingly, infusion of GLP-1produced an elevated glial reaction in the basal nucleus on the infusionside (FIG. 17F) than that apparent as a result of the lesion (FIG. 17E)or after vehicle infusion.

FIG. 18 shows percentage of difference in the Abercrombie correctednumber of ChAT-immunoreactive cell bodies in the ipsilateral basalnucleus (lesion side) relative to the intact contralateral basal nucleusin sham and ibotenic acid animals receiving intracerebroventricularly(i.c.v.) infusion of vehicle (artifical CSF: aCSF), exendin-4 or GLP-1.Vertical error bars represent the standard error of the differencebetween the means. Significant difference from ibotenic acid vehiclegroup; *p<0.05 and **p<0.01.

FIG. 19A, FIG. 19B, and FIG. 19C are bar charts showing that treatmentof PC 12 cells with GLP-1 and analogues significantly decreased βAPP andsAPP protein levels without cellular dysfunction. Treatment with NGF,exendin-4, exendin-4 WOT and GLP-1 was not associated with cellulardysfunction as determined by measurement of LDH levels from conditionedmedia samples compared with media standards (FIG. 19A). Densitometricquantification of the immunoprobed proteins are presented as the meanpercent change in expression of βAPP derivatives from cell lysatessamples (FIG. 19B) and soluble sAPP from conditioned media samples (FIG.19C) taken on day 3 of treatment relative to untreated control samplescultured in low serum media alone. The treatment conditions illustratedalong the x-axis are common to panels FIG. 19A, FIG. 19B and FIG. 19C.Vertical error bars represent standard error of 3 individualexperimental values. Significant difference from untreated: *p<0.05 and**p<0.01.

FIG. 20 shows GLP-1 treatment significantly reduced endogenous Aβ 1-40levels in control mice. Control mice were infused i.c.v. with GLP-1 (3.3μg and 6.6 μg), exendin-4 (0.2 μg), NGF (2 μg) and control (vehicle).Biochemical analysis of whole brain homogenates was carried out bysandwich ELISA for Aβ 1-40. Aβ values are expressed as the mean Aβconcentration in fmol/g±SEM from treated and untreated animals.Significant difference from control: **p<0.01.

FIG. 21 shows dose response curves for some of the Ex-4 and GLP-1 Gly8analogs. Intracellular cAMP levels were measured in Rin 1046-38 cellsafter treatment with the indicated concentrations of the peptides for 30min at 37 C. The data are normalized to maximum values obtained in eachexperiment for each peptide.

FIG. 22A and FIG. 22B are line graphs showing the acute biologicaleffects of the peptides on blood glucoselevels. The results withEx(1-36) (circle) and Ex(1-35) (square) are shown in FIG. 22A, and theresults with additional peptides are shown FIG. 22B. Blood glucose andinsulin levels were determined after an sc injection of 10 nmol/kg ofeach of the peptides to Zucker rats. The results are mean±SEM (n=4/groupfor FIG. 22A and n=3 for FIG. 22B).

FIG. 23 shows the abdominal fat volume lost over 51 days in control(white bars) and Ex (1-30) treated animals (black bars). The values areexpressed as a percentage of the initial total fat volume (0 days). Foreach group, the total fat lost, as well as the fat lost from thevisceral and subcutaneous tissue fractions is shown. The data shows thatthere was a significantly greater volume of total fat and visceral fatlost in the Ex(1-30) treated animals than in the control animals. Bothgroups showed decreased total fat volume at the 51 day time point. Inthe control group, this loss was largely due to loss of fat from thesubcutaneous fraction, which occurred to a similar extent in the treatedanimals. *P<0.05 Ex(1-30) vs control.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to specific synthetic methods, specifictreatment regimens, or to particular purification procedures, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” 30“an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “apolypeptide” includes mixtures of polypeptides, reference to “apharmaceutical carrier” includes mixtures of two or more such carriers,and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment 5includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. As used herein, “about” refers to the given value±10%.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

As used throughout, by “subject” is meant an individual. Preferably, thesubject is a mammal such as a primate, and, more preferably, a human.Thus, the “subject” can include domesticated animals, such as cats,dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).

The term “polypeptide” is used synonymously herein with the term“peptide.” Both “polypeptide” and “peptide” include a series ofnaturally occurring or non-naturally occurring amino acids connected bypeptide bonds.

By “isolated polypeptide” or “purified polypeptide” is meant apolypeptide that is substantially free from the materials with which thepolypeptide is normally associated in nature or in culture. Thepolypeptides of the invention can be obtained, for example, byextraction from a natural source if available (for example, a mammaliancell), by expression of a recombinant nucleic acid encoding thepolypeptide (for example, in a cell or in a cell-free translationsystem), or by chemically synthesizing the polypeptide. In addition,polypeptide may be obtained by cleaving full length polypeptides. Whenthe polypeptide is a fragment of a larger naturally occurringpolypeptide, the isolated polypeptide is shorter than and excludes thefull-length, naturally-occurring polypeptide of which it is a fragment.

The invention relates to novel polypeptide analogues of GLP-1 andexendin-4. As used herein, “GLP-1” is used synonymously with GLP-1 7-36amide, the amidated 5form of residues 7-36 of the complete GLP-1sequence, and GLP-1 7-37. Residues of exendin-4 are aligned with GLP-1,residues 7-36, and numbered according to the numbering of the GLP-1residues. Such a residue numbering convention is used throughout. SeeFIG. 1A-FIG. 1C.

The polypeptides, in a preferred embodiment, are insulinotropic. By“insulinotropic” is meant that the polypeptides increase insulinsynthesis, release or secretion 1.5, 2.0, 2.5, 3.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0 17.5, 18.0, 18.5,19.0, 19.5, or 20.0 times greater than basal release. The increase ininsulin release can be shown directly (e.g., by showing increased levelsof insulin) or indirectly (e.g., by showing reduced levels of glucose orby showing increased levels of cAMP) either in vivo (e.g., by assayingblood glucose levels) or in vitro (e.g., by assaying the level ofinsulin in the culture medium) using assay methods known in the art.

Insulinotropic effects can be due to any one of several mechanisms,including, for example, an increase in the number of insulin positivecells. The insulinotropic polypeptides, for example, promote insulinrelease by promoting differentiation of stem cells into insulin-positivecells and by promoting de-differentiation of non-stem cells to a lessdifferentiated state and then promoting differentiation intoinsulin-positive cells. As a second example, the insulinotropic effectsmay be caused by an increase in the amount of insulin synthesized and/orreleased by each insulin positive cell in a given period of time.Combined insulinotropic effects could also occur if the number ofinsulin positive cells is increased and the amount of insulin secretedby each cell is also increased.

By “basal release” is meant the amount of insulin released in responseto a glucose stimulus in the absence of a second releasing agent.

By “insulin-positive cells” is meant any cells that have been shown torelease insulin, including, for example, pancreatic islet cells, such asbeta cells, or cell lines such as RIN 1048-36 cells, any cells designedto release insulin (e.g., genetically modified cells that containinsulin); or any cells that contain insulin.

By “analogue of GLP-1 or exendin-4” is meant modified GLP-1 and exendinamino acid sequences that show agonist properties (i.e., show one ormore biological activities of GLP-1 or exendin-4). Such modificationsinclude chimeric polypeptides that include one or more amino acidresidues present in GLP-1 and one or more amino acid residues present inexendin-4. The modifications also include truncations of either GLP-1 orexendin-4 or the chimeric polypeptides. For example, a truncatedchimeric polypeptide is exendin-4 7-36 with the G at position 36replaced with the R in position 36 of GLP-1. The polypeptides of thepresent invention include one or more additional amino acids (i.e.,insertions or additions), deletions of amino acids, or substitutions inthe amino acid sequence of GLP-1 or exendin-4 without appreciable lossof functional activity as compared to GLP-1 or exendin-4. For example,the deletion can consist of amino acids that are not essential to thepresently defined differentiating activity and the substitution(s) canbe conservative (i.e., basic, hydrophilic, or hydrophobic amino acidssubstituted for the same) or non-conservative. Thus, it is understoodthat, where desired, modifications and changes may be made in the aminoacid sequence of GLP-1 and exendin-4, and a protein having likecharacteristics still obtained. Various changes may be made in the aminoacid sequence of the GLP-1 or exendin-4 amino acid sequence (orunderlying nucleic acid sequence) without appreciable loss of biologicalutility or activity and possibly with an increase in such utility oractivity.

The term “fragments” or “truncations” as used herein regarding GLP-1 orexendin-4 or polypeptides having amino acid sequences substantiallyhomologous thereto means a polypeptide sequence of at least 5 contiguousamino acids of either GLP-1, exendin 4, or polypeptides having aminoacid sequences substantially homologous thereto, wherein the polypeptidesequence has an insulinotropic function.

Other modifications include D-enantiomers, in which at least onenaturally occurring L-configuration of an amino acid residue is replacedby the D-configuration of the amino acid residue.

The present invention contemplates the use of a spacer, such as alateral spacer. The term “lateral spacer” is defined as a compound thatis incorporated within the amino acid sequence by chemical bonds,whereby the compound increases the distance between two or more aminoacid residues in order to reduce or eliminate the cleavage (e.g., by DPP1V) of the amino acid sequence at or near that position. For example, inthe sequence A-X-B, where A and B are amino acid residues and X is thelateral spacer, cleavage of the sequence by an enzyme is reduced oreliminated when compared to the sequence in the absence of the lateralspacer (A-B). Preferably 1 to 4 compounds can be incorporated into theamino acid sequence as the lateral spacer. Thus, 1, 2, 3, or 4 compoundsare inserted in various embodiments.

In general, the lateral spacer is any compound that can form a peptidebond with an amino acid, i.e., contains at least one amino group and atleast one carboxyl group (CO₂), where the carboxyl group can be acarboxylic acid or the ester or salt thereof. In one embodiment, thelateral spacer has the formula H₂N—R¹—CO₂H (I), wherein R¹ comprises asubstituted or unsubstituted, branched or straight chain C₁ to C₂₀ alkylgroup, alkenyl group, or alkynyl group; a substituted or unsubstitutedC₃ to C₈ cycloalkyl group; a substituted or unsubstituted C₆ to C₂₀ arylgroup; or substituted or unsubstituted C₄ to C₂₀ heteroaryl group. Inanother embodiment, R¹ can be represented by the formula (CH₂)_(n),where n is from 1 to 10. In a preferred embodiment, R¹ is (CH₂)₃(3-aminopropionic acid) or (CH₂)₅ (6-aminohexanoic acid).

The present invention provides a purified polypeptide, wherein thepolypeptide comprises a modified GLP-1 or exendin-4 sequence, or ananlogue thereof, with a spacer between the amino acid residuescomparable to residues 7 and 8 (designated in the case of GLP-1 with aAha spacer, for example, “GLP-1 Aha⁸”) or residues 8 and 9 (designatedin the case of GLP-1 with a Aha spacer, for example, “GLP-1Aha⁹”) ofGLP-1. The lateral spacer, in one embodiment, is one or moreaminoproprionic acid residues. In one embodiment, the spacer is a6-aminohexanoic acid spacer and the 6-aminohexanoic acid spacercomprises less than four 6-aminohexanoic acid residues. The polypeptide,for example, can comprise GLP-1 7-36 with one or more 6-aminohexanoicacid residues between residues 7 and 8 (i.e., GLP-1 Aha⁸) or cancomprise GLP-1 7-36 with one or more 6-aminohexanoic acid residuesbetween residues 8 and 9. The polypeptide can comprise GLP-1 7-36 withtwo or more 6-aminohexanoic acid residues between residues 7 and 8(i.e., GLP-1 Aha⁸) or can comprise GLP-1 7-36 with two or more6-aminohexanoic acid residues between residues 8 and 9. The polypeptide,for example, can comprise GLP-1 7-36 with three or more 6-aminohexanoicacid residues between residues 7 and 8 (i.e., GLP-1 Aha⁸) or cancomprise GLP-1 7-36 with three or more 6-aminohexanoic acid residuesbetween residues 8 and 9. More specifically, in one embodiment thepolypeptide comprises the amino acid sequence of SEQ ID NO:8, SEQ IDNO:22, or SEQ ID NO:23. In other embodiments, the polypeptide comprisesthe amino acid sequence of SEQ ID NO:42, SEQ ID NO:43, SEQ 1D NO:44, SEQID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO: 48, or SEQ ID NO:49. Inalternative embodiments, the polypeptide comprises the amino acidsequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:25, SEQ IDNO:33, wherein the amino acid sequence contains a spacer between theamino acid residues comparable to residues 7 and 8 or to residues 8 and9 of GLP-1.

In a preferred embodiment, the polypeptide of the present invention hasan insulinotropic effect that is comparable to the effect of anequimolar amount of GLP-1 or, in a more preferred embodiment, aninsulinotropic effect that is comparable to the effect of an equimolaramount of exendin-4. By “comparable to the effect” is meant an effectthat is within about 10-15% of the effect of GLP-1 or exendin-4. In aneven more preferred embodiment, the polypeptide has an insulinotropiceffect that exceeds the insulinotropic effect of either GLP-1 orexendin-4. By “exceeding the effect” of GLP-1 or exendin-4 is meant anincrease in insulinotropic effect compared to GLP-1 or exendin-4,preferably an increase that is greater than about 10% of the effect ofGLP-1 or exendin-4. Thus, in a preferred embodiment, the polypeptide ofthe present invention is as potent as GLP-1 or exendin-4, and in a morepreferred embodiment is more potent that GLP-1 and, optionally, morepotent than exendin-4.

In a preferred embodiment, the polypeptide of the present invention islonger acting than GLP-1. In a more preferred embodiment, thepolypeptide is at least as long acting as exendin-4. In an even morepreferred embodiment, the polypeptide is longer acting than exendin-4.By “longer acting” is meant that the polypeptide is more resistant thanGLP-1 or exendin-4 to at least one degradative enzyme. For example, thepreferred embodiment of the polypeptide of the present invention is moreresistant to degradation by the enzyme dipeptidyl dipeptidase (DPP1V)than is GLP-1 and, optionally, more resistant than exendin-4. Suchresistance to one or more degradative enzymes can be assessed directlyby detecting the amount of degradation products (e.g., the amount ofN-terminal degradation products) or the amount of un-cleavedpolypeptide. Alternatively, the resistance to one or more degradativeenzymes can be detected indirectly by assessing the reduction ininsulinotropic effect over time following administration of apolypeptide of the invention. For example, as the degradative enzymescleave the polypeptides of the invention, plasma insulin levels shoulddecline after a single administration. In a preferred embodiment thisdecline would be slower than for GLP-1 and perhaps even slower than forexendin-4.

In a preferred embodiment, the polypeptide has reduced antigenicity ascompared to exendin-4. Antigenicity can be assessed using routinemethods, such as biological assays designed to assess neutralizingantibodies and polypeptide clearance.

In a preferred embodiment, the polypeptide has a higher binding affinityfor the GLP-1 receptor than the binding affinity of GLP-1 for the GLP-1receptor. In a more preferred embodiment, the polypeptide has a higherbinding affinity for the GLP-1 receptor than the binding affinity ofexendin-4 for the GLP-1 receptor.

In a preferred embodiment, the polypeptide stimulates intracellular cAMPlevels over basal levels more than GLP-1. In an even more preferredembodiment, the polypeptide stimulates intracellular cAMP levels overbasal levels more than exendin-4.

Specifically, the invention provides a purified polypeptide, the aminoacid sequence of which comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:25, SEQ ID NO:33. More specifically, the invention provides apurified polypeptide, the amino acid sequence of which consistsessentially of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:25, SEQ IDNO:33. Even more specifically, the invention provides a purifiedpolypeptide, the amino acid sequence of which consists of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:33.

Also, the invention provides a purified polypeptide, the amino acidsequence of which comprises SEQ ID NO:3, 4, 16, 17, 18, 19, 20, 21, 22,23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or 41.More specifically, the invention provides a purified polypeptide, theamino acid sequence of which consists essentially of SEQ ID NO:3, 4, 16,17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36,37, 38, 39, 40, or 41. Even more specifically, the invention provides apurified polypeptide, the amino acid sequence of which consists of SEQID NO:3, 4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31,32, 34, 35, 36, 37, 38, 39, 40, or 41.

The polypeptides of the invention can be prepared using any of a numberof chemical polypeptide synthesis techniques well known to those ofordinary skill in the art including solution methods and solid phasemethods. Solid phase synthesis in which the C-terminal amino acid of thepolypeptide sequence is attached to an insoluble support followed bysequential addition of the remaining amino acids in the sequence is onesynthetic method for preparing the polypeptides. Techniques for solidphase synthesis are described by Merrifield et al., J. Am. Chem. Soc.85:2149-2156 (1963). Many automated systems for performing solid phasepeptide synthesis are commercially available.

Solid phase synthesis is started from the carboxy-terminal end (i.e.,the C-terminus) of the polypeptide by coupling a protected amino acidvia its carboxyl group to a suitable solid support. The solid supportused is not a critical feature provided that it is capable of binding tothe carboxyl group while remaining substantially inert to the reagentsutilized in the peptide synthesis procedure. For example, a startingmaterial can be prepared by attaching an amino-protected amino acid viaa benzyl ester linkage to a chloromethylated resin or a hydroxymethylresin or via an amide bond to a benzhydrylamine (BHA) resin orp-methylbenzhydrylamine (MBHA) resin. Materials suitable for use assolid supports are well known to those of skill in the art and include,but are not limited to, the following: halomethyl resins, such aschloromethyl resin or bromomethyl resin; hydroxymethyl resins; phenolresins, such as 4-(a-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxyresin; tert-alkyloxycarbonyl-hydrazidated resins; and the like. Suchresins are commercially available and their methods of preparation areknown to those of ordinary skill in the art.

The acid form of the peptides may be prepared by the solid phase peptidesynthesis procedure using a benzyl ester resin as a solid support. Thecorresponding amides may be produced by using benzhydrylamine ormethylbenzhydrylamine resin as the solid support. Those skilled in theart will recognize that when the BHA or MBHA resin is used, treatmentwith anhydrous hydrofluoric acid to cleave the peptide from the solidsupport produces a peptide having a terminal amide group.

The α-amino group of each amino acid used in the synthesis should beprotected during the coupling reaction to prevent side reactionsinvolving the reactive α-amino function. Certain amino acids alsocontain reactive side-chain functional groups (e.g. sulfhydryl, amino,carboxyl, hydroxyl, etc.) which must also be protected with appropriateprotecting groups to prevent chemical reactions from occurring at thosesites during the peptide synthesis. Protecting groups are well known tothose of skill in the art. See, for example, The Peptides: Analysis,Synthesis, Biology, Vol. 3: Protection of Functional Groups in PeptideSynthesis (Gross and Meienhofer (eds.), Academic Press, N.Y. (1981)).

A properly selected α-amino protecting group will render the α-aminofunction inert during the coupling reaction, will be readily removableafter coupling under conditions that will not remove side chainprotecting groups, will not alter the structure of the peptide fragment,and will prevent racemization upon activation immediately prior tocoupling. Similarly, side-chain protecting groups must be chosen torender the side chain functional group inert during the synthesis, mustbe stable under the conditions used to remove the α-amino protectinggroup, and must be removable after completion of the peptide synthesisunder conditions that will not alter the structure of the peptide.

Coupling of the amino acids may be accomplished by a variety oftechniques known to those of skill in the art. Typical approachesinvolve either the conversion of the amino acid to a derivative thatwill render the carboxyl group more susceptible to reaction with thefree N-terminal amino group of the peptide fragment, or use of asuitable coupling agent such as, for example,N,N′-dicyclohexylcarbodimide (DCC) or N,N′-diisopropylcarbodiimide(DIPCDI). Frequently, hydroxybenzotriazole (HOBt) is employed as acatalyst in these coupling reactions.

Generally, synthesis of the peptide is commenced by first coupling theC-terminal amino acid, which is protected at the N-amino position by aprotecting group such as fluorenylmethyloxycarbonyl (Fmoc), to a solidsupport. Prior to coupling of Fmoc-Asn, the Fmoc residue has to beremoved from the polymer. Fmoc-Asn can, for example, be coupled to the4-(a-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxy resin usingN,N′-dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole (HOBt) atabout 25° C. for about two hours with stirring. Following the couplingof the Fmoc-protected amino acid to the resin support, the α-aminoprotecting group is removed using 20% piperidine in DMF at roomtemperature.

After removal of the α-amino protecting group, the remainingFmoc-protected amino acids are coupled stepwise in the desired order.Appropriately protected amino acids are commercially available from anumber of suppliers (e.g., Novartis (Switzerland) or Bachem (Torrance,Calif.)). As an alternative to the stepwise addition of individual aminoacids, appropriately protected peptide fragments consisting of more thanone amino acid may also be coupled to the “growing” peptide. Selectionof an appropriate coupling reagent, as explained above, is well known tothose of skill in the art.

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in excess and the coupling is carried out in amedium of dimethylformamide (DMF), methylene chloride (CH₂Cl₂), ormixtures thereof. If coupling is incomplete, the coupling reaction maybe repeated before deprotection of the N-amino group and addition of thenext amino acid. Coupling efficiency may be monitored by a number ofmeans well known to those of skill in the art. A preferred method ofmonitoring coupling efficiency is by the ninhydrin reaction. Peptidesynthesis reactions may be performed automatically using a number ofcommercially available peptide synthesizers such as the Biosearch 9500™synthesizer (Biosearch, San Raphael, Calif.).

The peptide can be cleaved and the protecting groups removed by stirringthe insoluble carrier or solid support in anhydrous, liquid hydrogenfluoride (HF) in the presence of anisole and dimethylsulfide at about 0°C. for about 20 to 90 minutes, preferably 60 minutes; by bubblinghydrogen bromide (HBr) continuously through a 1 mg/10 mL suspension ofthe resin in trifluoroacetic acid (TFA) for 60 to 360 minutes at aboutroom temperature, depending on the protecting groups selected; or byincubating the solid support inside the reaction column used for thesolid phase synthesis with 90% trifluoroacetic acid, 5% water and 5%triethylsilane for about 30 to 60 minutes. Other deprotection methodswell known to those of skill in the art may also be used.

The peptides can be isolated and purified from the reaction mixture bymeans of peptide purification well known to those of skill in the art.For example, the peptides may be purified using known chromatographicprocedures such as reverse phase HPLC, gel permeation, ion exchange,size exclusion, affinity, partition, or countercurrent distribution.

The polypeptides of the invention can also be prepared by other meansincluding, for example, recombinant techniques. Examples of appropriatecloning and sequencing techniques, and instructions sufficient to directpersons of skill through many cloning exercises are found in Sambrook etal. (1989) Molecular Cloning-A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook).

The invention further provides a method of treating a subject withdiabetes, comprising administering to the subject the polypeptide of theinvention in an amount that has an insulinotropic effect. By “diabetes”is meant diabetes mellitus. The method of the present invention isconsidered to be useful in the treatment of a subject having type 2diabetes. The method of the present invention could be of use in otherforms of diabetes (including, for example, type 1 diabetes) when thepolypeptide promotes non-insulin producing cells to produce insulin.

The polypeptides of the present invention also have uses in the nervoussystem. In one embodiment, the polypeptides are neurotrophic (i.e.promoting proliferation, 5differentiation or neurite outgrowth) orneuroprotective (i.e. rescuing neuron cells or reducing neuronal celldeath). Thus, the invention further relates to a method of reducingneuronal death, comprising contacting one or more neurons with apolypeptide comprising GLP-1, exendin-4, or a neuroprotective orneurotrophic GLP-1 or exendin-4 analogue. Neuronal death may occur, forexample, with mechanical injury (e.g., trauma or surgery), toxic injury,neurodegenerative disease, apoptosis, and peripheral neuropathy. Oneskilled in the art would recognize that rescuing neurons (i.e.,promoting viability of cells that show signs of cell death) and reducingneuronal death (i.e., promoting viability of cells that do not showsigns of cell death) may be desired. For example, treatment with acompound that reduced neuronal death would be useful in treating anexplant or culture of neuronal cells, prior to subsequenttransplantation. Also, such treatment could be used to rescue neuronsand reduce neuronal death following a stroke, brain or spinal cordinjury, nerve injury, or neurotoxic injury. Furthermore, rescuingneurons or reducing neuronal death would be useful in the treatment ofneurodegenerative condition or disease diseases, including, for example,Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, multiple sclerosis, and peripheralneuropathy.

The invention also relates to a method of promoting neuronaldifferentiation or proliferation, comprising contacting one or moreneurons or neuronal precursor cells with a polypeptide comprising GLP-1,exendin-4, or a differentiation-inducing or proliferation-inducing GLP-1or exendin-4 analogue. Differentiation involves a transition from a cellstate in which the cell lacks neuronal characteristics (e.g., lackscharacteristics such as a distinct nucleolus, neuronal processes,extensive rough endoplasmic reticulum, expression of neuronal markers)to a cell state characterized by a neuronal phenotype. By neuronalproliferation is meant that stem cells or cells of neuronal lineagedivide and/or differentiate into neurons. The effect of eitherdifferentiation or proliferation is an increase in the number ofneurons. By “an increase in the number of neurons” is meant an additionof neurons to the total number of all neurons present. Thus, the rate ofneuronal cell death may exceed the rate of differentiation orproliferation, but the addition of new neurons is still considered to bean increase over the total neurons and such an increase in number, evenin the absence of an increase in the total number of living neurons,could still have therapeutic advantages.

The present invention also relates to a method of reducing formation oraccumulation of amyloid β protein, comprising contacting one or moreneurons with a polypeptide comprising GLP-1, exendin-4, or a GLP-1 orexendin-4 analogue that affects β-amyloid precursor protein metabolism.Such a method could be useful in lowering levels of amyloid protein orin preventing the deposition of amyloid protein, which is observed insenile plaques in a subject with Alzheimer's Disease. The method of thepresent invention could reduce formation or accumulation of amyloid βprotein by acting at various points in the processing β-amyloidprecursor protein. For example, the polypeptide may decrease synthesisof β-amyloid precursor protein, promote cleavage of β-amyloid precursorprotein within the amyloid β protein region, increase secretion ofsoluble β-amyloid precursor protein, decrease secretion of amyloid βprotein, or increase degradation of amyloid β protein.

The present invention also relates to a method of promoting growth ofneuronal processes, comprising contacting one or more neurons with apolypeptide comprising GLP-1, exendin-4, or a process-promoting GLP-1 orexendin-4 analogue. By “growth of neuronal processes” is meant either anincrease in the number of neuronal processes off of the soma, anincrease in the complexity of neuronal processes (usually due to anincrease in the number of branch points of axons or dendrites) or anincrease in length of the processes. The growth of neuronal processesmay be desired in many contexts, including for example, following aperipheral nerve injury or an injury to the central nervous system whereoptimization of regenerative capacity is desired. Also, inneurodegenerative conditions, the existing neurons may be able tocompensate for neruonal death with an enriched field of processes.

The present invention also relates to a method of treating a subjectwith a neurodegenerative condition or of reducing one or more symptomsof a neurodegenerative condition in a subject, comprising administeringto the subject a therapeutically effective amount of a polypeptidecomprising GLP-1, exendin-4, or a therapeutically effective GLP-1 orexendin-4 analogue. More specifically, the treatment could be directedto neurodegenerative conditions selected from the group consisting ofAlzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, stroke, multiple sclerosis, brain injury,spinal cord injury, and peripheral neuropathy.

Also, provided is a method of treating a subject with a neurotoxicinjury or of reducing one or more symptoms of a neurotoxic injury in asubject, comprising administering to the subject a therapeuticallyeffective amount of a polypeptide comprising GLP-1, exendin-4, or atherapeutically effective GLP-1 or exendin-4 analogue. Suchadministration could be before during or after the exposure to theneurotoxin. Neurotoxins include the neurotoxic form of amyloidβ-peptide, camptothecin, glutamate, etoposide, anti-cancer drugs, vincaalkaloids, 3-nitrognognonic acid, MPTP, domoic acid, kainic acid, andibotenic acid.

The contacting step in these neural methods is performed in vivo or invitro depending upon the desired effect. For example, neurons in culturecan be treated prior to or after manipulation in culture that mightcause neuronal death. Also, neurons in situ in the nervous system can betreated prior to or after exposure to a trigger that causes neuronaldeath. In a transplant paradigm, for example, the donor neurons to betransplanted might be treated in culture and then the transplantationarea of the brain or spinal cord can be treated to prevent neuronaldeath of the recipient's neurons and of the transplanted neurons.

The polypeptides related to uses in the nervous system includepolypeptides comprising GLP-1, exendin-4, and their biologically activeanalogues or agonists. Preferably, the analogues bind and activate theGLP-1/exendin-4 receptor. The polypeptides include for examplepolypeptides having the amino acid sequence of SEQ ID NO:1, 2, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 25, 33, 42, 43, 44, 45, 46, 47, or 48.Other examples include polypeptides having the amino acid sequence ofSEQ ID NO:3, 4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30,31, 32, 34, 35, 36, 37, 38, 39, 40, or 41.

Also provided by the present invention is a pharmaceutical compositioncomprised of a polypeptide of the invention, including for example,GLP-1, exendin-4, and their biologically active analogues or agonists,in combination with a pharmaceutically acceptable carrier.

One skilled in the art would recognize how to monitor the effectivenessof the treatment and how to adjust the treatment accordingly. Forexample, blood glucose levels could be monitored with normoglycemiabeing the optimal effect of treatment. If blood glucose levels arehigher than preferred levels, then the amount of polypeptideadministered should be increased, and, if blood glucose levels are lowerthan preferred levels, then the amount of polypeptide administered wouldbe decreased.

The dosages of the polypeptides to be used in the in vivo method of theinvention preferably range from about 0.1 pmoles/kg/minute to about 100nmoles/kg/minute for continuous administration and from about 0.01nmoles/kg to about 400 nmoles/kg for bolus injection. Preferably, thedosage of the polypeptide in in vivo methods range from about 0.01nmoles/kg/min to about 10 nmoles/kg/min. The exact amount required willvary from polypeptide to polypeptide and subject to subject, dependingon the species, age, and general condition of the subject, the severityof disease that is being treated, the particular polypeptide used, itsmode of administration, and the like. Thus, it is not possible tospecify an exact “insulinotropic amount” or an amount useful in treatingneuronal disease or injury. However, an appropriate amount may bedetermined by one of ordinary skill in the art using only routineexperimentation.

The polypeptides may be conveniently formulated into pharmaceuticalcompositions composed of one or more of the compounds in associationwith a pharmaceutically acceptable carrier. The compounds may beadministered orally, intravenously, intramuscularly, intraperitoneally,topically, transdermally, locally, systemically, intraventricularly,intracerebrally, subdurally, or intrathecally. One skilled in the artwould know to modify the mode of administration, the pharmacologiccarrier, or other parameters to optimize the insulinotropic effects. Theamount of active compound administered will, of course, be dependent onthe subject being treated, the subject's weight, the manner ofadministration and the judgment of the prescribing physician.

Depending on the intended mode of administration, the pharmaceuticalcompositions may be in the form of solid, semi-solid or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, suspensions, lotions, creams, gels, or the like,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include, as noted above, aneffective amount of the selected drug in combination with apharmaceutically acceptable carrier and, in addition, may include othermedicinal agents, pharmaceutical agents, carriers, adjuvants, diluents,etc. See, e.g., Remington's Pharmaceutical Sciences, latest edition, byE. W. Martin Mack Pub. Co., Easton, Pa., which discloses typicalcarriers and conventional methods of preparing pharmaceuticalcompositions that may be used in conjunction with the preparation offormulations of the polypeptides and which is incorporated by referenceherein.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc., an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example see Remington's PharmaceuticalSciences, referenced above.

For oral administration, fine powders or granules may contain diluting,dispersing, and/or surface active agents, and may be presented in wateror in a syrup, in capsules or sachets in the dry state, or in anonaqueous solution or suspension wherein suspending agents may beincluded, in tablets wherein binders and lubricants may be included, orin a suspension in water or a syrup. Where desirable or necessary,flavoring, preserving, suspending, thickening, or emulsifying agents maybe included. Tablets and granules are preferred oral administrationforms, and these may be coated. Parental administration, if used, isgenerally characterized by injection.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parental administration involves use of aslow release or sustained release system, such that a constant level ofdosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which isincorporated by reference herein.

For topical administration, liquids, suspension, lotions, creams, gelsor the like may be used as long as the active compound can be deliveredto the surface of the skin.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Peptide Design and Synthesis

A series of chimeric peptides was designed that incorporated fundamentalfeatures of exendin-4 and GLP-1. The sequences of the 35 peptides areshown in FIG. 1A, FIG. 1B, and FIG. 1C, along with the sequence forGLP-1 (residues 7-36) and exendin-4 (residues 7-45 numbered according tothe alignment of exendin-4 with the numbered GLP-1 residues). They weredesigned to (i) minimize the cleavage action of DDP1V between aminoacids 8 and 9, (ii) assess the minimal requirement for insulinotropicaction, and (iii) to assess which amino acid differences betweenexendin-4 and GLP-1 account for the former's 13-fold increase in potencyversus GLP-1. The peptides shown in FIG. 1A, FIG. 1B, and FIG. 1Cutilized L- and D-amino acids in their synthesis.

Peptides were synthesized on a PEG-Polystyrene resin usingFmoc-derivatives of amino acids in a Applied Biosystems (Foster, Calif.)automated peptide synthesizer using piperidine-dimethyl formamide fordeprotection and HOBt/HBTU for coupling. The finished peptides werecleaved from the resin using trifluoroacetic acid (TFA), precipitatedwith ether and subjected to purification using reverse phase HPLC on aC-18 hydrophobic resin in 0.1% TFA using an acetonitrile gradient. Thepurity of the final material was verified using reverse phase HPLC andthe mass of the peptide was verified using mass spectrometry. Allpeptides were of 95% or greater purity.

Other peptides were designed to reduce cleavage by DPP1V using 2-aminohexanoic acid (6-aminohexanoic acid). See Table 2.

TABLE 2 Aha-Containing Peptides Peptide No. Sequence. SEQ ID NO 45H/Aha/AEGTFTSDVSSYLEGQAAKEFIAWLVKG SEQ ID NO: 42 RPSSGAPPPS 46H/Aha/AEGTFTSDVSSYLEGQAAKEFIAWLVKG SEQ ID NO: 43 RPSSGAPPPSGAP 47H/Aha/AEGTFTSDVSSYLEGQAAKEFIAWLVKG SEQ ID NO: 44 RPSSGAPPPSGAPPSS 48HA/Aha/EGTFTSDVSSYLEGQAAKEFIAWLVKG SEQ ID NO: 45 RPSSGAPPPSHA/Aha/EGTFTSDVSSYLEGQAAKEFIAWLVKG SEQ ID NO: 46 RPSSGAPPPSGAPHA/Aha/EGTFTSDVSSYLEGQAAKEFIAWLVKG SEQ ID NO: 47 RPSSGAPPPSGAPPSS 49Y/Aha/AEGTFISDYSIAMDKIHQQDFVNVVLLAQ SEQ ID NO: 48 KGKKNDWKHNITQ 25HA/(Aha)₄/EGTFTSDVSSYLEGQAAKEFIAWLVK SEQ ID NO: 22 GLP-1(Aha⁹)₄ GR 26HA/(Aha)₈/EGTFTSDVSSYLEGQAAKEFIAWLVK SEQ ID NO: 23 GLP-1(Aha⁹)₈ GR

Example 2 Insulin Secretion In Vitro

RIN 1048-36 cells, a gift from Dr. Samuel A. Clark (Bio HybridTechnologies, Shrewsbury, Mass.) were used to monitor the action ofGLP-1, exendin-4 and analogs on insulin secretion. Cells were seeded ata density of 2.5×10⁵ cells/cm² on glass coverslips placed on the bottomof 12-well dishes and grown for 48 h. Thereafter, they were preincubatedfor 2 periods of 30 min each with glucose-free buffer (containing mM:140 NaCl, 5 KCl, 1 NaPO4, 1 MgSO4, 2 CaCl, 20 HEPES buffer (pH 7.4), and0.1% bovine serum albumin) in a 37° C. humidified incubator. Thereafter,cells were incubated for 1 h at 37° C. in the presence of 1 mL of thesame buffer with 5 mM glucose and peptide (1×10−8 M). GLP-1 andexendin-4 (1×10-8 M) were used as standards in all assays. After 1 h themedium was removed and stored at −80° C. prior to quantification ofinsulin levels by EIA (Crystal Chem, Chicago Ill.), and the cells werelysed with HCl (300 μl, 0.1 M, 20 min, RT) for measurement of totalprotein using the Bradford method (Bio-Rad, Richmond, Calif.) withbovine γ-globulin as a standard.

As shown in FIG. 2, some of the amino acid modifications induced insulinsecretion in a manner comparable to or exceeding inducement by GLP-1 orexendin-4. Several modifications were used to reduce recognition byDDP1V of the cleavage site between amino acid residues 8 and 9. Thereplacement of L-amino acids with the D-form near amino acid residues 8and 9, however, proved ineffective, as shown with peptides 2, 3, and5-7, because the peptides were incapable of inducing insulin secretion.Peptide 4 (not shown in FIG. 1A, FIG. 1B, and FIG. 1C), the GLP-1sequence with residues 7-14 being D-amino acids, was similarly incapableof inducing insulin secretion. When an amino acid spacer wasincorporated before or between residues 8 and 9, peptide 11 (SEQ IDNO:8) (having the 4 amino acid spacer before residue 8) potently inducedinsulin secretion whereas peptide 25 (SEQ ID NO: 22) (having a 4 aminoacid spacer between residues 8 and 9) and peptide 26 (SEQ ID NO:23)(having an 8 amino acid spacer between residues 8 and 9) did not induceinsulin secretion. Replacement of amino acid 8 (alanine:A) in GLP-1 witha small neutral amino acid, the peptide in the comparable position inexendin-4 (i.e., glycine:G) induced insulin secretion slightly more thanexendin-4. See GLP-1 Gly⁸ (SEQ ID NO:3).

Additional substitutions of the GLP-lamino acid residues with exendin-4residues resulted in peptides that retained the ability to induceinsulin secretion. For example, peptide 8 (SEQ ID NO:5) (having an A

G substitution at position 8 and a V

L substitution at position 16), peptide 9 (SEQ ID NO: 6) (having A

G, V

L, S

K, Y

Q, and L

M substitutions at position 8, 16, 18, 19, and 20, respectively),peptide 10 (Ex-WOT; SEQ ID NO:7) (having the same substitutions as inpeptide 9 and additional having G

E, Q

E, A

V, K

R, E

L, A

E, V

K, K

N, and R

G substitutions at residues 22, 23, 25, 26, 27, 30, 33, 34, 36,respectively) all retained the ability to induce insulin secretion. Infact, peptide 8 (SEQ ID NO:5) had a substantially greater effect oninsulin secretion than either GLP-1 or exendin-4.

The addition of the terminal 8-9 amino acids present on exendin-4 ontoGLP-1, as in peptide 12 (SEQ ID NO:9), resulted in a peptide that alsohad a substantially greater effect on insulin secretion than eitherGLP-1 or exendin-4. When residue 8 in exendin-4 (i.e., glycine:G) wassubstituted with residue 8 of GLP-1 (i.e., alanine:A) and the terminal 9amino acids of exendin-4 were retained, as in peptide 13 (SEQ ID NO:10),or removed, as in peptide 14 (SEQ ID NO:11), then both peptides retainedthe ability to induce insulin secretion; however, peptide 13 had asubstantially greater effect than exendin-4 without the modifications.

Truncations of exendin-4 were also tested for their ability to induceinsulin secretion. See peptides 15-24 (SEQ ID NOs:12-21). Only thosepeptides including more than 32 residues (i.e., peptides 15-18 (SEQ IDNOs:12-15)) induced insulin secretion. Of those truncation peptides thatinduced insulin secretion, peptide 15 (SEQ ID NO:12) (including residuesup to and including residue 43) and peptide 18 (SEQ ID NO:15) (includingresidues up to and including residue 34) were the only ones that had aninducing effect that exceeded that of exendin-4 or GLP-1.

Modifications designed to affect the charge of the peptide were alsoundertaken. GLP-1 bears a net neutral charge, possessing a total of 4+charges related to basic amino acids at positions 7, 26, 34 and 36, and4− charges related to acidic amino acids at positions 9, 15, 21 and 27.Exendin-4 bears a net negative charge related to a basic domain atposition 21-23. Exendin-4 possesses a total of 4+ charges (positions 7,18, 26, 33) and 6− charges (positions 9, 15, 21, 22, 23, 30), whereasits 9 amino acid terminal tail is neutral. The addition of a singlebasic amino acid (providing a positive charge) for the tail of exendin-4(i.e., the replacement of small neutral glycine, G, by larger arginine,R, in position 36), as in peptide 34 (SEQ ID NO:31), results ininactivity of the peptide in in vitro insulin secretion. Arginine, R, iswell tolerated in position 36 of GLP-1, and when retained or replaced inGLP-1 by neutral glycine, G, and the exendin-4 tail (peptide 12(SEQ IDNO:9) it remains active and actually has an activity that exceeds thatof exendin-4. Interestingly, arginine, R, is well tolerated in position36 of exendin-4 when position 30 is modified from acidic glutamic acid,E, to neutral alanine, A (peptide 36 (SEQ ID NO:33)) when position 27bears a negative charge. Also, the replacement of a neutral serine, S,by a basic lysine, K, to introduce a positive charge into position 18(peptide 1 (SEQ ID NO:3) as compared to peptide 30 (SEQ ID NO:27)),results in a loss of activity; however, the replacement of neighboring(position 19, 20) neutral amino acids, tyrosine, Y, and leucine, L, byneutral amino acids, glutamine, Q, and methionine, M, restores activity(peptide 9 (SEQ ID NO:6) as compared to peptide 30 (SEQ ID NO:27)).

When the insulin secreting effects of GLP-1 and the 6-arninohexanoicacid-containing peptides were compared, GLP-1 Aha⁸ (peptide 11; SEQ IDNO:8) was shown to be as effective as GLP-1. GLP-1 Aha⁸ (peptide 11; SEQID NO:8) induced insulin secretion about 1.2-fold above basal levels.See FIG. 3. The insertion of additional 6-aminohexanoic acid residues,however, as in peptides 25 and 26 (SEQ 1D NOs:22-23), abrogated thepeptides' ability to induce insulin secretion.

Example 3 Intracellular cAMP Determination

CHO cells stably transfected with the human GLP-1 receptor, GLP-1Rcells, were grown to 60-70% confluency on 12-well plates, washed threetimes with Krebs-Ringer phosphate buffer (KRP), and incubated with 1 mlof KRP containing 0.1% bovine serum albumin (BSA) for 2 h at 37° C. in ahumidified air incubator. Cells were then incubated in 1 ml of KRPsupplemented with 0.1% BSA with Isobutylmethylxanthine (IBMX) (1 mM;Calbiochem, La Jolla, Calif.) in the presence or absence of the peptidesunder study. The reaction was stopped 30 min later by washing the intactcells three times with ice-cold phosphate buffered saline (PBS). Theintracellular cAMP was extracted by incubating the cells in ice-coldperchloric acid (0.6 M, 1 ml, 5 min). After adjusting the pH of thesamples to 7 using potassium carbonate (5 M, 84 μI), sample tubes werevortexed and the precipitate formed was sedimented by centrifugation (5min, 2000×g, 4° C.). The supernatant was vacuum-dried and solubilized in0.05 M Tris (pH 7.5) containing 4 mM EDTA, (300 μl). Sodium carbonate(0.15 μM) and zinc sulfate (0.15 μM) were added, to the samples whichwere then incubated for 15 min on ice. The resulting salt precipitatewas removed by centrifugation (5 min, 2000×g, 4° C.). The samples wereassayed in duplicate aliquots (50 μl) using a [³H] cAMP competitivebinding assay kit (Amersham, Philadelphia Pa.).

Levels of cAMP were measured in cells treated with GLP-1, the6-aminohexanoic acid-containing peptides, or the D-amino acid containingpeptides. Intracellular cAMP levels generated by the GLP-1 analogs wereassessed initially at a peptide concentration of 1 OnM (theconcentration at which maximum cAMP production is seen with GLP-1). Thedata are shown in FIG. 4. The peptides were incubated with theCHO/GLP-1R cells in the presence of IBMX for 30 min at 37° C. Inagreement with the results from the in vitro insulin assay, the D-aminoacid substitutions throughout the GLP-1 molecule resulted in only asmall increase above basal levels—i.e., those obtained with IBMX alone.Also, GLP-1 (Aha⁹)₄ (SEQ ID NO:22) and GLP-1 (Aha ⁹)₈ (SEQ ID NO:23)were inactive when compared to the insulinotropic compounds, GLP-1 Gly⁸(SEQ ID NO:3) and GLP-1 Aha⁸ (SEQ ID NO:8). The induction of cAMP inresponse to varying concentrations of GLP-1, GLP-1 Gly⁸, or GLP-1 Aha⁸was measured. See FIG. 5. Table 3 shows the ED50 values of all threecompounds. GLP-1 Aha⁸ (0.5 nM) stimulated intracellular cAMP productionto 4-fold above basal it however exhibited a higher ED50 when comparedto GLP-1 and GLP-1 Gly⁸.

TABLE 3 IC₅₀ and EC₅₀ Values Derived from the Binding and cAmpExperiments, Respectively Peptide Name IC₅₀ (nM)^(a) EC₅₀ (nM) GLP-1 3.7 ± 0.2 0.036 ± 0.002 GLP-1 Gly⁸ 41 ± 9 0.13 ± 0.02 GLP-1 Aha⁸ 22 ± 70.58 ± 0.03 GLP-1 (Aha⁹)₄ 236 ± 25 ND GLP-1 (Aha⁹)₈ 400 ± 34 ND GLP-1 D3301 ± 40 ND GLP-1 D5 350 ± 20 ND GLP-1 D8  265 ± 115 ND GLP-1 D12  574 ±216 ND GLP-1 D21 ND ND GLP-1 All D ND ND ^(a)The concentration thatreached 50% of ¹²⁵I-GLP-1 binding was calculated in three to fourseparate experiments performed in triplicate.

Example 4 Competitive Binding of Peptides to GLP-1 Receptor in IntactCells

Binding studies were performed in the manner of Montrose-Rafizadeh etal. (1997b, J. Biol. Chem. 272:21201-206. Briefly CHO/GLP-1R cells weregrown to confluency on 12-well plates and washed with serum-free HamF-12 medium for 2 h before the experiment. After two washes in 0.5 mlbinding buffer (10), cells were incubated overnight at 4 C with 0.5 mlbuffer containing 2% BSA, 17 mg/L Diprotin A (Bachem, Torence, Calif.),10 mM glucose, 1-1000 nM GLP-1 or other peptides and 30,000 cpm SI-GLP-1(Amersham, Philadelphia, Pa.). At the end of the incubation thesupernatant was discarded, and the cells were washed three times withice-cold PBS and incubated at room temperature with 0.5 ml of 0.5N NaOHand 0.1% sodium dodecyl sulfate for 10 min. Radioactivity in celllysates was measured in an ICN Apec-Series g-counter. Specific bindingwas determined as total binding minus the radioactivity associated withcells incubated in the presence of a large excess of unlabeled GLP-1 (1μM).

The potential of these GLP-1 analogs to displace [¹²⁵1] GLP-1 by bindingcompetitively to the human GLP-1 receptor was then examined. CHO/GLP-1Rcells were incubated with [¹²⁵1] labeled GLP-1 in the absence andpresence of varying concentrations of the peptides. See FIG. 6A-FIG. 6C.

The IC50 values obtained for those compounds which bound competitivelyto the GLP-1 receptor are shown in Table 3. Insertion of the6-aminohexanoic acid moiety resulted in a reduction in binding to thereceptor. With the increase in the length of the spacer 6-aminohexanoicacid groups in the 9-position, there was a dramatic decrease in affinityfor the GLP-1 receptor. The lack of biological activity seen with theD-amino acid substituted compounds can be explained by their markedlyreduced ability to bind to the GLP-1 receptor. There was a progressivereduction in receptor recognition with increasing D-amino acidsubstitution such that compounds GLP-1 D21 (peptide 6) and GLP-1 All D(peptide 7) did not displace the labeled GLP-1.

Example 5 Acute In Vivo Activity

The acute maximal insulin response was determined by quantifying plasmainsulin levels in Zucker rats following intravenous peptideadministration. Specifically, following overnight fasting, diabetic malerats, approximately 400 g weight, were anesthetized with 50 mg/kgpentobarbital and a catheter was tied into their right femoral arteryfor blood collection. Thereafter, a bolus of exendin-4, GLP-1 or peptide(0.4 nmol/kg) was administered into their left saphenous vein over 30 s(N=6 per peptide). Blood, taken prior to peptide administration and at5, 15, 30, 60 and 90 min thereafter, was drawn into heparinized tubescontaining EDTA and aprotinin for insulin determination. Plasma wasseparated, removed and immediately frozen to −70° C. The insulin levelsthen were quantified by using a rat insulin ELISA kit (Crystal ChemInc., Chicago, Ill.).

The acute in vivo activity of two examples of potent peptides from thein vitro studies above, peptide No. 10 (Ex-4 WOT; SEQ ID NO:7) andpeptidel (GLP-1 Gly⁸; SEQ ID NO:3), was assessed in fasted, diabeticZucker rats to induce insulin secretion. Peak plasma insulinconcentrations are shown in FIG. 7 following equimolar administration ofpeptides (0.4 nmol/kg) and are compared to those achieved afterequimolar exendin-4 and GLP-1. Both Ex-4 WOT and Gly-8 potentlyincreased plasma insulin concentrations.

As illustrated in FIG. 7, the in vitro action of peptides to induceinsulin secretion in RIN 1048-36 cells correlates with in vivo activityto acutely elevate plasma insulin concentrations in fasted diabeticZucker rats, as exemplified by peptide 1 (GLP-1 Gly⁸; SEQ ID NO:3) andpeptide 10 (Ex-4 WOT; SEQ ID NO:7) after their i.v. administration. Ofparticular note is the finding that Ex-4 WOT), which lacks the terminal9 amino acids of exendin-4, proved to be more potent than did equimolarexendin-4. Similarly, peptide 1 (GLP-1 Gly ⁸) proved to be more potentthan equimolar GLP-1.

Example 6 Duration of In Vivo Activity

The time-dependent duration of insulinotropic action was evaluated byquantifying plasma insulin and glucose levels in Zucker rats followingintraperitoneal (i.p.) peptide administration. Specifically, afterovernight fasting, diabetic male rats, approximately 400 g weight, wereanesthetized with 50 mg/kg pentobarbital and a catheter was tied intotheir right femoral artery for blood collection. Thereafter, a bolus ofexendin-4, GLP-1 or peptide (0.4 nmol/kg) was administered i.p. (1≧2 perpeptide). Blood, taken prior to peptide administration, at 30 and 60min, and at 2, 4, 6 and 24 h, was drawn into heparinized tubescontaining EDTA and aprotinin for insulin determination, and a separatesample was taken to measure glucose. Plasma was separated, removed andimmediately frozen to −70° C. Thereafter insulin levels were quantifiedby using a rat insulin ELISA kit (Crystal Chem Inc., Chicago, Ill.) andplasma glucose was quantified by the glucose oxidase method.

As shown in FIG. 8, specific amino acids modifications provide a longduration of action on in vivo insulin. In this regard, the action ofpolypeptide 10 (Ex-4 WOT; SEQ ID NO:7) on plasma insulin levels provedto be long acting, like exendin-4. In addition, similar to acutestudies, polypeptide 10 proved to be more potent than equimolarexendin-4 in diabetic rats. In contrast, polypeptide 1 (GLP-1 Gly⁸; SEQID NO:3) and polypeptide 11 (GLP-1 Aha^(b), SEQ ID NO:8) proved to havean action on the time-dependent insulin response that was intermediatebetween GLP-1 and exendin-4; being longer than the former but shorterthan the latter. In addition, similar to acute studies, polypeptides 1and 11 proved to be more potent than equimolar GLP-1 in diabetic rats.

Example 7 MALDI Mass Spectroscopy

GLP-1 (2 μM) and GLP-1 Aha⁸ (SEQ ID NO:8) (2 μM) were incubated with 5mU recombinant DPP1V (Calbiochem, La Jolla, Calif.) in PBS for 10 minand 2 h respectively at 37° C. Both compounds (100 μM, 100 μl ) wereincubated in an equivalent volume of human serum at 37° C. for 2 h. Inall cases, enzymatic reactions were quenched by the addition oftrifluoroacetic acid (0.1% v/v final concentration). Samples wereimmediately analyzed using Matrix Assisted Linear DesorptionIonisation-Time Of Flight (MALDI-TOF) mass spectrometry. A MicromassMALDI-TOF (Micromass, Beverly, Mass.) reflectron instrument was used ata laser energy of 15-25% over a mass range of 1000-6000 Da, with 5 lasershots summed per spectrum. Alpha-cyano-4-hydroxycinnamicacid (Sigma, St.Louis, Mo.) was used as a matrix and was prepared to a concentration of10 mg/ml in a 8 mg/ml ammonium carbonate (Sigma, St. Louis, Mo.) buffer.One microlitre samples were diluted 50/50 v/v with matrix before beingtransferred to the MALDI plate.

The stability of GLP-1 Aha⁸ (SEQ ID NO:8) was compared with GLP-1 in thepresence of DPP1V and human serum. Treatment of GLP-1 (2 μM) with DPP1V(5 mU) for 10 min or 100% serum for 2 h at 37° C. caused a considerableincrease in the amount of N-terminal truncated product (Mr=3089 gmol−1)as measured by MALDI. In contrast GLP-1 Aha⁸ (2 μM) appeared resistantto either treatment.

Example 8 Determination of Biological Activity of GLP-1Aha⁸ In Vivo

Six-month old male Zucker fa/fa rats (Harlan, Indianapolis, Ind.) andsix-month old Wistar rats were used in this study. They were allowed adlibitum access to chow and water and were on a 12 h light, 12 h darkcycle (lights on 0700 h). The bedding for the Zucker rats was a paperbased product, “Carefresh” (Absorption Co., Belingham, Wash.). TheZucker rats were fasted on wire, in the absence of bedding, overnightbefore the experiment. Wistar rats were fasted on their normal bedding.General anaesthesia was induced by an intraperitoneal injection ofpentobarbital (50 mg/kg). A cannula was placed in the femoral artery forblood sampling and the polypeptides (GLP-1 Aha⁸ (SEQ ID NO:8) and GLP-1Gly⁸ (SEQ ID NO:3), 24 nmol/kg) were injected subcutaneously into thenape of the animals' necks (n=5 for each treatment group) Blood glucoselevels were measured by the glucose oxidase method using a GlucometerElite (Bayer Corp. Diagnostics, Tarrytown, N.Y.).

To verify that GLP-1 Aha⁸ had biological activity in vivo, thepolypeptide was administered subcutaneously (24 nmol/kg) to fastedZucker fatty (fa/fa) and Wistar rats. Another group of Zucker ratsreceived a similar dose of GLP-1 Gly⁸. Blood glucose was then monitoredfor the next 8 h. In FIG. 9A and FIG. 9B, the results show that bothcompounds rapidly lowered blood glucose. In Zucker rats, the reductionin blood glucose was more pronounced with GLP-1 Gly⁸, due to the factthat the fasting glucose was lower in that group, but the slope andmagnitude of the decline was similar for both compounds. Insulinsecretion was attenuated in the GLP-1 Gly⁸ due to the drop in bloodglucose into the hypoglycemic range, proving the glucose-dependency ofinsulinotropism with this class of compounds. As the GLP-1 Aha⁸-treatedZucker rats did not become hypoglycemic the insulinotropic response didnot become abrogated and the prolonged effect can be seen. In Wistarrats, which are not hyperglycemic in the fasting state, insulin levelsincreased rapidly with GLP-1 Aha⁸, leading to hypoglycemia and againrapid attenuation of the insulinotropic response.

Example 9 Truncation of Exendin-4 and the Biological Importance of the9-Amino Acid C-Terminal Tail of Exendin-4

This study was performed to determine the importance of the nineC-terminal amino acids to the biological activity of Ex-4. A sequence oftruncated Ex-4 analogs and GLP-1 analogs to which the nine C-terminalsequence has been added were used in the study.

Materials and Cell Lines

Peptides were synthesized as described above. All peptides were of 95%or greater purity. Table 4 shows the sequences of the GLP-land exendin-4analogs studied. Isobutylmethylxanthine (IBMX) was purchased fromCalbiochem (La Jolla, Calif.). Exendin-4 and GLP-1-(7-36)amide wereobtained from Bachem (Torrance, Calif.). The cloned rat insulinoma cellline RIN 1046-38 was a gift from Dr. Samuel A. Clark (Bio HybridTechnologies, Shrewsbury, Mass.) and were routinely cultured in M199with Earle's salts (Mediatech, Inc., Herndon, Va.) suppplemented withglucose (11 mM), 50 U/ml penicillin, 50 μg/ml streptomycin, andglutamine (2 mM) in a humidified 5% CO₂-95% air incubator at 37 C.Chinese hamster ovary (CHO) cells stably transfected with the humanGLP-1 receptor, CHO/GLP-1R cells, were described above. Plasma insulinlevels were measured by ELISA (Crystal Chem Inc., Chicago Ill.). HbAlcwas measured as described in Greig et al., 1999. Blood glucose levelswere measured using a Glucometer Elite (Bayer Diagnostics, Tarrytown,N.Y.).

TABLE 4 The amino acid sequences of the GLP-1 and exendin-4 analogsstudied 7    11      16    21     20      25 36 GLP-1(7-36)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 1) GLP-1(7-36) GLP-1 ETHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRPSSGAPPPS Exending(31-39) (SEQ NO: 9)1     5      10     15     20     25 30    35 Exendin-4 Ex-4HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ NO: 2) Exendin (1-36)Ex(1-36) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAP (SEQ NO: 49) Exendin(1-35) Ex(1-35) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGA (SEQ NO: 13) Exendin(1-33) Ex(1-33) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSS (SEQ ID NO: 14)Exendin (1-30) (Ex- Ex(1-30) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG (SEQ ID 4WOT) NO: 11)  7    11    16    21      20      25 GLP-1Gly⁸(7-36) GGHGEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 3) GLP-1Gly⁸(7-36) GG1HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRPSSGAPPPS Exendin(31-39) (SEQ ID NO: 50)GLP-1Gly⁸(7-36) GG2 HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRPSSGAP Exendin(31-36)(SEQ ID NO: 51) GLP-1Gly⁸(7-36) GG3HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRPSS (SEQ Exendin(31-33) ID NO: 52)The underlined amino acids refer to those from the exendin-4 sequencethat are being studied.

Animals

Six-month-old male Zucker fa/fa rats (Harlan, Indianapolis, Ind.),2-month old C57BLKS/J-Leprdb/Leprdb mice (Jackson Laboratories, BarHaror, Mass.) and 2-month old Fisher rats (Harlan, Indianapolis, Ind.)were used in the acute and chronic experiments. All animals were allowedad libidum access to chow and water. Animals were on a 12 hourlight-dark cycle (lights on 7 am). The bedding for the Zucker rats anddb/db mice was a paper-based product, Carefresh (Absorption Co.,Belingham, Wash.) and the Fisher rats were housed on normal bedding.

Intracellular cAMP Determination

RIN 1046-38 cells, grown to 60-70% confluence on 12-well plates weretreated as described in Example 3 and cAMP determinations were performedaccordingly. Dose response curves are shown in FIG. 21.

Competitive Binding of Peptides to GLP-1 Receptor in Intact Cells

Binding studies were performed as described in Example 4. Table 5 showsIC₅₀ and EC₅₀ values derived from the competitive binding in CHO GLP-1Rcells and cAMP assays in RIN 1046-38 cells respectively.

TABLE 5 The IC₅₀ and EC₅₀ values derived from the competitive binding inCHO GLP-1R cells and cAMP assays in RIN 1046-38 cells respectively.Peptide Name IC₅₀(nM) EC₅₀(nM) GLP-1 44.9 ± 3.2 GG 220 ± 23 GG₁  74 ± 11GG₂ 129 ± 39 GG₃  34.5 ± 14.5 GLP-1 ET 21.2 ± 2.9 Ex-4 3.22 ± 0.9 Ex(1-36)  8.8 ± 1.4 ND Ex (1-35) 7.0 ± 2  ND Ex (1-33) 49.0 ± 1.1 ND Ex(1-30) 32.0 ± 5.8 Ex (1-28) 45.0 ± 5.7 Ex (1-26) No binding No activityEx (1-23) No binding No activity Ex (1-20) No binding No activity Ex(1-17) No binding No activity Ex (1-14) No binding No activity Ex (1-11)No binding No activity Ex (1-8) No binding No activity The concentrationthat reached 50% of [I^(I25)] GLP-1 binding was calculated in three orfour separate experiments performed in triplicate. Peptides of a few as11 amino acids were assessed and such amino acids had minimal binding.

Statistical Analysis

All values are shown as the mean±SEM, and the differences among thegroups were analyzed using ANOVA. The curves for FIG. 1A, FIG. 1B, andFIG. 1C and FIG. 2 were fitted with a four-parameter sigmoid logisticregression equation using an iterative computer program(20), and theEC50 and IC50 values in Table 2 were calculated from the fitted data.

Acute Time-Course Experiments in Zucker Fa/Fa Rats

Six-month-old male Zucker fa/fa rats (Harlan, Indianapolis, Ind.) wereused in this study as described above in Example 5, except 10 nmol/kg ofpeptide was administered in a PBS solution containing 0.1% BSA. Bloodglucose levels following sc injections of Ex (1-36) and Ex (1-35) areshown in FIG. 22A-FIG. 22B.

Chronic Study with Ex (1-30) in Db/Db Mice

Animals were housed in our facilities for 2.5 months to facilitate theiracclimatization before the experiment commenced. For the first 20 daysof treatment the animals were given Ex (1-30) (1 nmol/kg) byintraperitoneal (ip) injection daily at 9 am. Thereafter, animalsreceived ip Ex (1-30) (1 nmol/kg) at approximately 9 am and 9 pm for thefollowing 32 days. At the end of the first 20 days of the treatmentprotocol blood glucose levels and HbAlc were measured. Food intake andanimal weight was determined daily at the time of the 9 am injection. Noday was missed in the schedule. Magnetic resonance images (MRI) weretaken on 51 and an IPGTT was performed on day 52.

At day 20 the HbAlc were 7.8±0.4 for the Ex(1-30) treated mice and7.7±0.3 for the saline treated mice. The fasting blood glucose valueswere 412±92 mg/dl for the Ex(1-30) treated mice and 600±1 mg/dI for thesaline treated mice.

Magnetic Resonance Imaging

Magnetic resonance images were obtained using a 1.9 T, 31 cm bore BrukerBioSpec system (Broker Medizintechnik GmbH, Ettlingen, Germany), a 20 cminner diameter shielded gradient set and a 5 cm diameter volumeresonator. The animals used in the chronic study were placed underisofluorane anesthesia and standard T1 weighted multislice spin-echoimages (TR=500 ms, TE=8.5 ms) were obtained over 20 contiguoustransverse slices of thickness 2.1 mm each, covering a region whichincluded the entire abdomen. The field of view was 5×5 cm over 128×128pixels. Each image was acquired using 8 acquisitions, over a totalimaging time of approximately 9 minutes. Imaging was performed on allanimals at two time points (day 0 and day 51).

Separation of visceral and subcutaneous regions was performed (BrukerParavision software) by drawing regions of interest (ROIs) for eachslice. Segmentation of adipose from normal tissue was achieved usingintensity histograms derived from each ROI (NIH Image software, NationalInstitutes of Health, USA). The histograms generally showed twowell-separated peaks (corresponding to water and adipose tissue), whichwere isolated using the valley between them as the demarcation point,enabling the adipose tissue content in each ROI to be summed.

The results are shown in FIG. 23. Although both sets of animals (controland Ex(1-30)-treated) lost weight, the Ex(1-30) treated animals showed areduction in visceral fat deposition. The treated animals did not loseweight as fast as the controls, which did not receive the drug. Thus thetreatment alleviated the diabetes and the treated animals werehealthier.

Example 10 The Effect of GLP-1 and GLP-1 Analogues on the Metabolism ofβ-Amyloid Precursor Protein (βAPP)

One of the important pathological hallmarks of Alzheimer's disease (AD)is the cerebrovascular deposition of senile plaques comprised largely ofamyloid-β peptide (Aβ). AP is derived from the larger glycosylatedmembrane-bound protein β-amyloid precursor protein ((βAPP). The majorityof βAPP is proteolytically cleaved within the Aβ domain to generate asoluble derivative (sAPP), which prevents the formation of amyloidogenicfragments. This study was performed to determine the effect GLP-1 andtwo analogues on the processing of the β-amyloid precursor protein.

PC12 cells were cultured in RPMI 1640 supplemented with 10%heat-inactivated horse serum, 5% heat-inactivated fetal calf serum, 25mM Hepes buffer and 1× antibiotic-antimycotic solution (all culturemedia and sera were obtained from MediaTech Inc. (Herndon Va.).Treatments were carried out in low serum in the presence of GLP-1 (3.3,33, and 330 μg/ml) (Bachem, Torrance, Calif.), and two analogues,exendin-4 (0.1, 1.0 and 10 μg/ml) and exendin-4-WOT (peptide 10 (Ex-4WOT; SEQ ID NO:7)) (0.1 and 1.0 μg/ml). NGF (5, 10, 25 and 5Ong/ml)(Promega, Madison, Wis.), which has been shown to stimulate thesecretory pathway resulting in more sAPP being secreted by PC12 cellsinto the conditioned medium, was used as a positive control. Followingtreatment for three days, conditioned media and cell lysates fromuntreated (low serum medium alone) and treated cells were subjected toimmunoblot analysis using the monoclonal antibody, 22C11 (RocheMolecular Biochemicals, Indianapolis, Ind.). The antibody, raisedagainst E. Coli-made βAPP whose epitope region has been assigned toβAPP₆₆₋₈₁ in the ectoplasmic cysteine-containing domain, recognizes allmature forms of βAPP found in cell membranes, as well ascarboxy-truncated soluble forms secreted into conditioned media. Intypical immunoblots of conditioned media or cell lysates from treated oruntreated cells, multiple high molecular weight protein bands (M_(r)100-140 kDa) were evident. The differences observed in the profile ofimmunoreactive bands in the immunoblots was due neither to the unequalloading of proteins into the gel nor to the uneven transfer of proteinsonto the membrane. Equivalent amounts of total protein were loaded ineach lane of the gel and the efficiency of the electrophoretic transferwas monitored by staining the membranes with 0.1% Ponceau S in 5% aceticacid.

Densitometric quantification of the upper band revealed dramaticincreases in intracellular levels of βAPP following NGF treatment (FIG.11A, bars 1 and 2; FIG. 11B, bars 1 and 2). Inherent variation betweencell culture experiments accounts for the difference in the degree ofdifferentiation following treatment with 5 ng/ml and 10 ng/ml NGF in oneseries of studies (FIG. 11A and FIG. 11C), and with 25 ng/ml and 50ng/ml NGF in a different series of studies (FIG. 11B and FIG. 11D). Incontrast to NGF, GLP-1 and analogues decreased intracellular levels ofβAPP (FIG. 11A, bars 3-5; FIG. 11B, bars 3-7). The combination of NGFand exendin-4 increased intracellular βAPP relative to untreated cells,but at a level in between that of the two treatment conditions alone(FIG. 11A, bar 6).

As shown in FIG. 11C and FIG. 11D, all doses of nerve growth factortreatment resulted in dramatic increases in secreted, solublederivatives of βAPP which could be detected in the conditioned medium(FIG. 11C, bars 1 and 2; FIG. 11D, bars 1 and 2). Following a similarpattern to intracellular βAPP levels from cell lysates, treatment withall doses of GLP-1, exendin-4 and exendin-4-WOT, produced decreases indetectable levels of sAPP in conditioned media (FIG. 11, bars 3-5; FIG.11D, bars 3-7). The combination of NGF and exendin-4 did not produce anychange in sAPP levels relative to untreated cells (FIG. 11C, bar 6).

Using the lactate dehydrogenase (LDH) kit from Sigma Co., the assay ofLDH was performed as described below in the conditioned medium and celllysate samples of both treated and untreated cells that were used. Nosignificant change was observed in the level of LDH between treated anduntreated cells under the conditions used. The possibility of toxicityas a result of treatment with GLP-1 and analogues, at the doses used,can be ruled out.

The data indicate reduced levels of secreted derivatives and matureforms of βAPP following GLP-1 treatment in PC12 cells. These reductionsin sAPP secretion may be a consequence of reduced βAPP synthesis.

Example 11 GLP-1 and Analogues Promote Neuronal Proliferation andDifferentiation

The effects of GLP-1 and two of its long-acting analogues, exendin-4 andexendin-4 WOT, on neuronal proliferation and differentiation and on themetabolism of neuronal proteins in the rat pheochromocytoma (PC12) cellline were tested. GLP-1 and exendin-4 induced neurite outgrowth, whichwas reversed by co-incubation with the selective GLP-1 receptorantagonist, exendin (9-39). Furthermore, exendin-4 enhanced nerve growthfactor (NGF) initiated differentiation and rescued degenerating cellsfollowing NGF-mediated withdrawal.

Materials

7S NGF was purchased from Promega (Madison, Wis.). GLP-1 and exendin(9-39) were obtained from Bachem (Torrance, Calif.). Exendin-4 and itsanalogue exendin-4 WOT were synthesized and assessed to be >95% pure byHPLC analysis as described above. All other chemicals were of highpurity and obtained from Sigma Chemicals (St. Louis, Mo.), unlessotherwise stated.

Data Analysis

Statistical analyses were performed where appropriate. Results areexpressed as mean±SEM (where SEM=standard error of the differencebetween the means). Analysis of variance (ANOVA) was carried out usingSPSS version VII, where p<0.05 was considered statistically significant.Following significant main effects, planned comparisons were made usingTukey's Honestly Significant Difference test (Tukey's HSD).

Culture Conditions

Pheochromocytoma cells were obtained from Dr. D. K. Lahiri(Indianapolis) and RIN 1046-38 cells (a clonal rat insulinoma cell line)were a gift from Dr. Samuel A. Clark (Bio Hybrid Technology, Shrewsbury,Mass.). PC12 cells were cultured in RPMI 1640 supplemented with 10%heat-inactivated horse serum, 5% heat-inactivated fetal calf serum, 25mM Hepes buffer and 1× antibiotic-antimycotic solution. RIN 1046-38cells were grown in medium 199 containing 12 mM glucose and supplementedwith 5% heat-inactivated fetal calf serum, 0.03% glutamine, 50 U/mlpenicillin and 50 mg/ml streptomycin. Cell culture media and sera wereobtained from MediaTech (Cellgro), Inc.(Herndon, Va.). The cells weregrown in a humidified atmosphere containing 5.0% CO₂. They were seededat approximately 2.0×10⁶ cells per 60 mm dish. PC12 cells were grown oncultureware coated in rat-tail collagen (Roche Molecular Biochemicals,Indianapolis). The 7S NGF was prepared by dilution in growth media at aconcentration of 100 mg/ml and stored at −20° C. Stock solutions ofGLP-1 and analogues were made fresh in sterile water and stored at −20°C.

Three dishes for each treatment condition were prepared. Treatmentsbegan 24 hours after seeding, once cells were well attached. The mediumwas aspirated, and 3 ml of fresh low serum media (containing only 0.5%fetal calf serum) with the appropriate compound(s), added.

Preparation of Cell Lysates

Conditioned media and cell pellets were harvested daily for proteinanalysis by immunoblotting. Cell lysates were prepared as follows. Thecells from the plate were collected gently and centrifuged at 800 g for10 minutes. The cell pellet was suspended in lysis buffer containing 10mM Tris-HCl (pH 7.4), 1% SDS, 0.174 mg/ml phenylmethylsulfonyl fluoride(PMSF), 1 mg/ml each of aprotinin, leupeptin, pepstatin A, and 4 ml of amixture of 45.98 mg/ml sodium vanadate and 10.5 mg/ml sodium fluoride.The suspended cells were triturated and centrifuged at 14,000 g for 15minutes. The proteins of the supernatant solution (cell lysate) weremeasured (Bradford, 1976) and analysed by immunoblotting.

Protein Analysis by Western Blotting

Western blot analysis was performed on ten micrograms of protein fromeach cell lysate and conditioned media sample using 10% Tris-glycinegels containing 2.6% Bis-acrylamide (Novex, San Diego Calif.). Proteinswere blotted onto PVDF paper. Transferred proteins were visualized bystaining the membrane with 0.1% Ponceau S solution in 5% acetic acid(Sigma).

Exendin-4 and GLP-1 Mediated Neurite Outgrowth

PC12 cells were grown on 60 mm dishes as above and cultured for fourdays. During this time neurite outgrowth was quantified daily. Fiverandom fields of cells were evaluated per dish and the proportion ofneurite-bearing cells was determined. Approximately 100 cells per fieldwere scored for neurites equal to or greater in length than that of thecell body. A cell was only scored once, although it may have had morethan one process per cell.

PC12 cells, when grown in complete media without the presence ofneurotrophic compounds, displayed none of the characteristics ofneuronal cell types. When exposed to NGF in low serum medium, the cellsstopped dividing and developed morphological properties similar tosympathetic neurons. The cells extended long processes, some becominghighly branched with the cell body exhibiting a more flattenedappearance than in cells cultured in low serum medium alone.

Treatment with GLP-1 or exendin-4 in low serum medium produced similareffects on differentiation to those induced by NGF. GLP-1 and exendin-4induced neurites were generally shorter in length, and less branchedthan neurites generated following treatment with NGF. In contrast, theGLP-1 antagonist, exendin (9-39) in combination with exendin-4 failed toinitiate neurite extension.

Daily quantification of neuritic development was also performed. Theresults shown in FIG. 12 represent the counts taken on day 3 oftreatment and are expressed as a percentage of control untreated cells.Growing PC12 cells in the presence of low serum medium alone resulted in5-10% of the cells extending neuritic projections. Analysis revealed asignificant main effect of treatment condition (F=263.5, df=8.89,p<0.001). As expected, NGF treatment significantly induced the neuronalphenotype at the three doses tested here; 10, 30 and 100 ng/ml (allp<0.01). For example, the treatment of cells with 10 and 30 ng/ml NGFproduced 550 and 720% increase in neurite projections from controls,respectively. Under the same conditions when PC12 cells were treatedwith exendin-4, a significant neuritic outgrowth was also observed at 1μg/ml (98% increase relative to untreated, p<0.05) and 10 μg/ml (160%increase relative to untreated, p<0.01) of the compound. However, theneurite extension with exendin-4 was not as pronounced as that ofNGF-treated cells. To determine the synergistic effect of the twocompounds, a combination treatment paradigm was tried. When exendin-4(100 ng/ml) was co-treated with either NGF at 10 ng/ml or 30 ng/ml, asignificant increase in neurite outgrowth was observed relative tountreated control cells (596% and 819% increase respectively, bothp<0.01). Enhancement in neurite outgrowth relative to NGF treatmentalone was only significant at 30 ng/ml(p<0.01). Similar results wereobserved with other doses of exendin-4 either alone or in combinationwith NGF. These data suggest that exendin-4 can initiate differentiationand can enhance NGF-induced differentiation.

Effect of Exendin-4 on NGF-Mediated Cell Death

PC12 cells were grown in complete media (RPMI 1640+5% Fetal bovineserum+10% Horse serum) in the presence/absence of 50 ng/ml NGF or thepresence/absence of exendin-4 (1 or 5 mg/ml). Cells were harvested after4 or 7 days, and subsequently allowed to rejuvenate in regular media foran additional 3 days. On the final day, cells were harvested and a MTTassay was performed to determine the

proportion of viable cells. In a second series of experiments(prevention) cells were cultured in the presence of 50 ng/ml NGF andexendin-4 (1 or 5 mg/ml) for 4 or 7 days. Cells were harvested andallowed to rejuvenate as above. In a third series of experiments(rescue) cells were cultured in the presence of 50 ng/ml NGF for 4 days.Exendin-4 at 5 mg/ml was added to the media for an additional 3 days.Cells were harvested on day 7 and allowed to rejuvenate as above.

In a fourth series of experiments (rescue), cells were grown in thepresence of NGF. On day 4, 5 mg/ml exendin-4 was added for an additional3 days. Cells were harvested on day 7 and allowed to rejuvenate asabove. Cells were counted in each plate (4 plates/treatment condition)by the trypan blue exclusion method and MTT assays were performed onDays 4 and 7, as described below.

In these experiments NGF withdrawal after 4 days failed to cause massivecell death, and, largely, cells were capable of almost fullyrejuvenating. Exendin-4 co-treatment did not show significant effects.Withdrawal of NGF after 7 days of treatment caused a 15-20% reduction incell viability, and the cells were not capable of fully rejuvenating(FIG. 13, bar 2). In this case exendin-4 co-treatment did not preventcell death, at either the low (FIG. 13, bar 4) or the high (FIG. 13, bar6) dose, or when added after 4 days of NGF treatment (FIG. 13, bar 7).However, when exendin-4 treatment was carried out followingNGF-withdrawal, revival processes were enhanced. For example, when PC12cells were cultured in the presence of NGF for 4 days, NGF was withdrawnand exendin-4 added from days 4-7 (FIG. 13, bars 8 and 9), cellsurvivability reached control values (>95%). This was the case for boththe high (5 mg/ml) and the low (1 mg/ml) dose of exendin-4.

MTT Assay

The CellTiter 96® Aqueous One Solution Cell Proliferation Assay Reagentfrom Promega (Madison, Wis.) was used in a colourimetric procedure fordetermination of the number of viable cells in a modified MTT assay. TheReagent contains a novel tetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]and an electron coupling reagent (phenazine ethosulfate; PES). PES hasenhanced chemical stability, which allows combination with MTS to form astable solution. The MTS tetrazolium compound is bioreduced by cellsinto a coloured formazan product, which is soluble in tissue culturemedium. This conversion is presumably accomplished by NADPH or NADHproduced by dehydrogenase enzymes in metabolically active cells(Berridge and Tan 1993). Assays are performed by the addition of a smallamount of the Reagent directly to cultured wells, incubation for 1-4hours and subsequent absorbance at 490 nM with a 96-well plate reader.The quantity of formazan product, as measured by the amount of 490 nmabsorbance, is directly proportional to the number of living cells inculture. Since the MTS formazan product is soluble in tissue culturemedium the present procedure requires fewer steps than procedures thatuse tetrazolium components such as MTT.

Partial Inhibition of Neurite Outgrowth by a PKA Inhibitor

Differentiated cultures were treated with 50 μM PD98059 or 40 μMLY294002, which inhibit ERK MAPK and PI3-K, respectively, to determinethe mechanism of GLP-1 and exendin-4 induced neurite outgrowth. Todetermine whether cAMP-dependent MAPK phosphorylation was controlled byPKA, GLP-1 and NGF-induced neurites were treated with the PKA specificinhibitor, H89.

Specifically, the cultures were treated for 48 hours with the GLP-1antagonist, exendin (9-39); with the PI3 kinase inhibitor, LY294002 (40μM); with the MAP kinase inhibitor, PD98059 (50 μM) or with the PKAinhibitor, H89 (20 μM). Cells were seeded onto 60 mm dishes atapproximately 1×10⁵ cells/ml and treated with either 10 nM GLP-1 or 0.3μM exendin-4 with each of the aforementioned compounds. NGF at 50 ng/mland forskolin (PKA activator) (20 μM) were used as positive controls inthese treatments.

Both PD98059 and LY294002 reduced GLP-1 and exendin-4 induced neuriteoutgrowth of the cells. Similarly, NGF-induced neuritic extension wasreduced following PD98059 and LY294002 treatment. The involvement ofboth the ERK MAP kinase and the PI3 kinase signaling pathways is thusimplicated in GLP-1 and exendin-4 mediated neurite production in PC12cells. Treatment with H89 demonstrated some inhibitory effects on GLP-1and NGF-induced neurite outgrowth. These data suggest that PKA isinvolved in the regulation of the MAP kinase signaling pathway but othersignaling pathways are also involved.

Expression of Synaptophysin and Beta-2/NeuroD

To examine the molecular changes that are occurring during GLP-1,exendin-4, or exendin-WOT induced differentiation of PC12 cells, theprofile of synaptophysin, which is a 37 kDa phosphorylated protein wellexpressed in the synaptic vesicle membrane, was studied. Thesynaptophysin monoclonal antibody (Oncogene Research Products, San DiegoCalif.), which stains neurosecretory vesicles of PC12 cells, was used.The membranes were blocked with 20 mM Tris, 500 mM NaCl pH 7.4, 1% (w/v)casein (BioRad, San Diego Calif.) at 37° C. for 1 hour. Primary antibodywas diluted in block and incubated with the proteins overnight at 4° C.The membrane was vigorously washed with 20 mM Tris pH 7.4, 150 mM NaCland 0.05% Tween-20 (TBST), three times for 15 minutes at roomtemperature. The peroxidase-linked secondary antibody in block wasincubated with the membrane for 2 hours at room temperature.Peroxidase-linked anti-mouse IgM (Chemicon, Tenecula, Calif.) was usedas the secondary antibody against synaptophysin. Excess antibody waswashed off with three vigorous washes in TBST prior to incubation in ECLPlus (Amersham, Philadelphia, Pa.) for 5 minutes. The membrane wassubsequently exposed to photographic film. Densitometric quantificationof the protein bands was performed using Molecular Analyst software(BioRad, Hercules, Calif.).

Western immunoblot analysis of cell lysate samples using thesynaptophysin antibody revealed a molecular weight band of approximately37 kDa. Treatment with NGF, GLP-1 and GLP-1 analogues dramaticallyreduced the expression of the synaptophysin protein compared tountreated cells. Densitometric quantification of the protein bandsshowed significant reductions for all treatment conditions relative tountreated (FIG. 14, all p<0.01), which appeared to be dose-dependent. Noimmunoreactive band was detected in conditioned media samples from PC12cells.

The high degree of differentiation in PC12 cells as a result of NGFtreatment was accompanied by a marked decrease in synaptophysinexpression relative to untreated control cells. Nerve growth factordemonstrated dose related changes in cellular synaptophysin expression,producing an approximately 70% maximal decrease relative to controlcells. GLP-1 and analogues, which showed similar effects on neuriticextension to NGF-mediated differentiation but to a lesser degree, showedcomparatively smaller decreases in synaptophysin expression.Interestingly, NGF and exendin-4 in combination produced a largerdecrease in synaptophysin expression than either compound alone,reflecting the additive morphological effects (FIG. 14). Overall,exendin-4 showed a more pronounced induction of differentiation in PC12cells, in terms of synaptophysin expression, than did either GLP-1 orexendin-4 WOT.

To investigate the role of the transcription factor Beta-2/NeuroD inGLP-1 induced differentiation in PC12 cells, cell lysates were probedwith the NeuroD polyclonal antibody (Santa Cruz Biotechnology Inc.,Santa Cruz, Calif.). Beta-2/NeuroD plays a major role in both neuronaland pancreatic endocrine development. Expression of NeuroD appears to betransient in sensory and motor neurons of the peripheral nervous system,sensory organs as well as parts of the brain and spinal cord duringneuronal differentiation; however detection in the adult brain maysuggest a secondary role in mature neurons (Lee et al 1997). Beta-2expression in pancreatic endocrine cells, the intestine and the brain,activates insulin gene transcription and can induce neurons todifferentiate. Mutant mice lacking the functional Beta-2 gene have astriking reduction in the number of insulin-producing beta cells, failto develop mature islets and as a consequence develop severe diabetesoften resulting in perinatal death (Naya et al 1997). Thus,Beta-2/NeuroD is essential for in vivo pancreatic development andneuronal differentiation.

NeuroD production was determined by Western blot analysis usinganti-NeuroD antibody as described above for synaptophysin antibody,except peroxidase-linked anti-goat IgG (Santa Cruz Biotechnology Inc.)was used as the secondary antibody. A 43 kDa band, apparent in bothuntreated and GLP-1 treated PC12 cell lysates was detected, which wasincreased following GLP-1 treatment. Beta-2/NeuroD expression isincreased following treatment with GLP-1, providing further evidence forthe neuronal differentiation properties of this insulinotropicpolypeptide. As anticipated, cultures exposed to low serum medium aloneshowed nominal expression of Beta-2/NeuroD. Indeed, Noma et al (1999)have shown that overexpression of NeuroD in transfected PC12 cellsinduced morphological changes such as neurite-like processes andsynapse-like structures, without a differentiating-inducing agent suchas NGF.

Demonstration of GLP-1 Receptor Presence in PC12 Cells

PC12 cells were plated onto poly-L-lysine coated glass coverslips in 35mm culture dishes and grown under standard conditions (as describedabove). Cells were fixed with 0.25% glutaraldehyde for 30 minutes.Endogenous peroxidase activity was quenched with 0.3% H₂O₂ andincubation in the primary polyclonal antibody (dilution factor 1:1500)raised against the N-terminal of the GLP-1 receptor (a gift from Dr.Joel F. Habener, Massachusetts General Hospital, MA) was carried out atroom temperature for 1 hour. Visualization used the avidin-biotinperoxidase method with subsequent development in diarninobenzidinedihydrochloride (DAB) following incubation in the biotinylatedanti-rabbit IgG secondary antibody.

The presence of GLP-1R-positive immunoreactive staining in PC12 cellsconfirmed the presence of the GLP-1 receptor. More specifically,staining was on the cell body and to a lesser extent on the neuriteterminal. However, not all PC12 cells expressed positive immunoreactivestaining to the same degree, although almost all cells appearedpositive.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

RT-PCR was performed as a sensitive assay for GLP-1 receptor mRNA. Ratinsulinoma cells (RIN cells) were used as a positive control. Total RNAwas isolated from PC12 cells using the method of Chomczynski and Sacchi(1987). 2.5 mg RNA was used in our RT-PCR reaction. RT-PCR wasundertaken in a volume of 50 ml of buffer containing 50 mM KCl, 10 mMTris-HCl, 3.5 mM MgCl2), 200 mM dNTP's and 0.4 mM of each rat GLP-1Rsense (5′ ACAGGTCTCTTCTGCAACC 3′) and antisense (5′ AAGATGACTTCATGCGTGCC3′) oligonucleotide primers (5′- and 3′-ends of the pancreatic GLP-1receptor sequences). Amplification was undertaken for 30 cycles in thepresence of [a-32P]dCTP. Rat islet cells were used as the positivecontrol. RT-PCR products (10 ml) were separated on a 4-20%polyacrylamide gel with appropriate size markers. The gel wassubsequently dried under a vacuum at 80° C. for 1 hour and exposed toX-ray film.

RT-PCR products of the expected size for the GLP-1 receptor wereobtained. Clear bands at 928-bp in rat islet mRNA and PC12 cell mRNAconfirmed the presence of the GLP-1 receptor on PC12 cells.

cAMP Determination

Before cAMP determination, PC12 cells were treated with 33 μg/ml GLP-1for 3 days. Triplicate cultures were harvested at 5-minute intervalsafter the onset of treatment for a total period of 30 minutes. Cellsharvested at the start of treatment (zero minutes) were used forbaseline levels of cAMP. Cyclic AMP was measured according to the methodof Montrose-Rafizadeh et al (1997a).

Activation of the GLP-1 receptor has been shown to stimulate adenylylcyclase, leading to an increase in intracellular cAMP. Cyclic AMP wasassayed over 30 minutes following treatment of PC12 cells with 33 mg/mIGLP-1. There was a maximal 1200-fold increase in cAMP levels within 15minutes of stimulation, which returned to near baseline within 30minutes. These findings demonstrate the presence and activity of theGLP-1 receptor on PC12 cells.

Toxicity Assay

The potentially toxic effects of exendin-4 were tested in vitro by twomethods: LDH assay and trypan blue exclusion method. The LDH assay wasperformed using a Sigma kit. Conditioned media samples collected atdifferent time intervals following treatments were subjected to asensitive lactate dehydrogenase (LDH) assay. The LDH assay provided ameasure of the number of cells via total cytoplasmic LDH or by membraneintegrity as a function of the amount of cytoplasmic LDH released intothe medium. The measurement of released LDH was based on the reductionof NAD by the action of LDH. The resulting reduced NAD (NADH) wasutilized in the stoichiometric conversion of a tetrazolium dye. Thefinal coloured compound was measured spectrophotometrically. If thecells were lysed prior to assaying the medium, an increase or decreasein cell number resulted in a concomitant change in the amount ofsubstrate converted. This indicated the degree of cytolysis or membranedamage (cytotoxicity) caused by the test material.

There were no significant changes in viable cell numbers followingtreatment, suggesting our compounds were not toxic to PC12 cells underthe conditions studied. See FIG. 15. To determine the integrity of thecell membrane during treatment, LDH levels were measured in theconditioned medium from control and treated cells under the sameconditions on day 3, in two separate series of experiments. As expected,LDH levels were elevated relative to the media standards (samples weretaken at the start of treatment). However, with the sole exception of 10ng/ml NGF and 10 μg/ml exendin-4, no dose of any treatment significantlyelevated LDH levels beyond control untreated cells. Exendin-4 at 10μg/ml elevated levels to 1.65-fold that of controls (p<0.01) and 10ng/ml NGF showed a 1.38-fold increase (p<0.05).

Cell Turnover in PC 12 Cells Determined by Incorporation ofBromodeoxyuridine

To determine whether GLP-1 affects the proliferation of PC12 cells inculture, cell proliferation in low serum medium was assessed bymonitoring incorporation of 5′-bromo-2′-deoxy-uridine (BrdU).Immunocytochemistry with an anti-BrdU antibody after labeling was usedto identify cells that were actively replicating DNA at the time oflabeling. PC12 cells were cultured for 3 days in the presence or absenceof 33 μg/ml GLP-1 or 50 ng/ml NGF. 10 μM BrdU was added to the culturemedium for 6 hours prior to fixing in 4% paraformaldehyde, to labelcellular DNA. The remainder of the method was followed according to theproliferation kit (Roche, Indianapolis, Ind.). Proliferating cells(those that were undergoing DNA replication at the time of BrdUlabeling) exhibited dark-staining nuclei with the chromagen reaction.BrdU incorporation was quantitated on days 1, 2 and 3 of treatment.Three dishes for each treatment condition were counted and expressed asthe percentage of labeled cells relative to the total number of cells.PC12 cells showed increased incorporation of BrdU on day 1 followingtreatment with NGF (9% increase relative to untreated) and GLP-1 (18%relative to untreated).

Example 12 Protection and Reversal of Excitotoxic Neuronal Damage byGlucagon-Like Peptide-1 and Exendin-4

The ability of GLP-1 and its long-acting analogue, exendin-4, to protectcultured hippocampal neurons against cell death induced by glutamate,and to attenuate ibotenic acid-induced cholinergic marker deficit inadult rats was tested.

Culture Conditions

Hippocampal neuronal cultures were prepared from 18-day-old embryonicSprague Dawley rats using methods similar to those described previously(Mattson et al., 1995). Briefly, cells were dissociated by mildtrypsination and trituration and plated in Minimal Essential Mediumcontaining 10% FBS and 1% antibiotic solution (10⁴ U/ml penicillin G, 10mg/ml streptomycin and 25 μg/ml amphotericin B; Sigma Chemicals, St.Louis, Mo.). Hippocampal neurons were plated at a density of 100,000cells/ml on 25 mm diameter poly-D-lysine coated glass coverslips. Threehours after plating the media was replaced with serum-free Neurobasalmedium containing 1% B-27 supplement (Gibco/Life Technologies, Carlsbad,Calif.).

Immunofluorescence staining for MAP-2 (neurons) and GFAP (astrocytes)showed that more than 98% of the cells were neurons and the remainderwere predominantly astrocytes. Cultures were used within 7-10 days ofplating.

Binding Studies

Binding studies were performed as described by Montrose-Rafizadeh(1997b). Duplicate hippocampal neuronal cultures were washed in 0.5 mlbinding buffer and subsequently incubated in 0.5 ml buffer containing 2%BSA, 17 mg/liter diprotin A (Bachem, Torrance, Calif.), 10 mM glucose,0.001-1000 nM GLP-1 and 30,000 cpm ¹²⁵I-GLP-1 (Amersham PharmaciaBiotech, Little Chalfont, UK), overnight at 4° C. At the end of theincubation the supernatant was discarded, and the cells washed threetimes in ice-cold PBS and incubated at room temperature with 0.5 ml 10.5M NaOH and 0.1% SDS for 10 min. Radioactivity in cell lysates wasmeasured in an Apec-Series γ-counter (ICN Biomedicals, Inc., Costa Mesa,Calif.). Specific binding was determined as the total binding minus theradioactivity associated with cells incubated in the presence of a largeexcess of unlabelled GLP-1 (1 μM). The GLP-1 concentration associatedwith 50% binding, EC₅₀, was determined by logit plot analysis.

Binding of ¹²⁵I-GLP-1 to cultured hippocampal neurons was displaced,concentration-dependently, by unlabelled GLP-1 (FIG. 16A). Theconcentration of GLP-1 required to displace 50% bound ¹²⁵I-GLP-1 wasdetermined by logit plot analysis and required a concentration of 14 nMGLP-1 (r=−0.999) in cultured hippocampal neurons.

cAMP Determination

To demonstrate presence of functional GLP-1 receptors, cyclic AMP wasmeasured according to the method of Montrose-Rafizadeh et al., (1997a).Triplicate hippocampal neuronal cell cultures were treated with 10 nMGLP-1 and harvested at 5-minute intervals after the onset of drugtreatment for a total period of 30 minutes. Cells harvested at the startof drug treatment (zero minutes) were used for baseline levels of cAMP.

Treatment of cultured hippocampal neurons with 10 nM GLP-1 evoked anincrease in cAMP production (FIG. 16B). There was a maximal two- tothree-fold increase in cAMP levels within 15 minutes of stimulation,which returned to near baseline within 30 minutes. One-way ANOVAdemonstrated significant main effects (F=9.45, df=6.20, p<0.001) oftreatment on cAMP production. Subsequent multiple comparisons usingTukey's HSD test revealed significant increases in cAMP production after10 (p<0.01) and 15 (p<0.001) min. These data demonstrate that primaryhippocampal neurons express functional GLP-1 receptors, making them anappropriate in vitro system in which to study potential protective andtrophic effects of these peptides.

Apoptotic Cell Death

The fluorescent DNA binding dye Hoescht 33342 was used to measureapoptotic cell death. Neurons were incubated in Locke's buffer withGLP-1 (10 nM) or exendin-4 (0.3 μM) in the presence of absence ofglutamate (10 μM) for 16 h. The concentration of GLP-1 used was based onthe EC50 value derived from the binding experiment, which wasdemonstrated to stimulate the release of cAMP, and which induceddifferentiation without causing cell death in our previous neuronal cellstudies. Cells were fixed in a solution of 4% paraformaldehyde in PBSand membranes were permeabilized with 0.2% Trition X-100. Followingincubation with Hoechst 33342 (1 μM) for 30 min, nuclei were visualizedunder epifluorescence illumination (340 nm excitation, 510 nm barrierfilter) using a 40× oil-immersion objective. Approximately 200 cellswere counted in at least three separate dishes for each treatmentcondition, and experiments were repeated at least twice. Cells wereconsidered apoptotic if nuclear DNA was fragmented or condensed, whereascells with nuclear DNA of a more diffuse and uniform distribution, wereconsidered viable. At the time of counting, the investigator was unawareof the identity of the treatment groups. The percentage of cells withcondensed or fragmented nuclei was determined in each culture.

Primary hippocampal neurons were treated overnight with 10 μM glutamate.Post-fixation, the cells were stained with Hoechst 33342, and the numberof apoptotic cells counted. In cells cultured in medium alone, 23% ofthe neurons exhibited apoptotic nuclei. Glutamate treatment produced 73%apoptosis (FIG. 16C). Concurrent treatment with either 10 nM GLP-1 (24%apoptotic cells) or 0.3 μM exendin-4 (25% apoptotic cells) completelyprotected against the cell death (FIG. 16B). Treatment with GLP-1 orexendin-4 alone did not produce any increase in the percentage ofapoptotic cells (20% and 23%, respectively) beyond that of controllevels. The values represent the pooled means of six individualexperiments. The percentage of cells undergoing apoptosis as a result ofeach treatment condition were subjected to ANOVA using StatViewstatistical software (Cary, N.C.). Following significant main effects, aposteriori comparisons of treatment vs. control were made using Tukey'sHonestly Significant Difference (HSD) test, using the pooled ANOVA errorterm and degrees of freedom. One-way ANOVA demonstrated statisticallysignificant differences in the extent of cell death between each insult(F=35.31, df=5.36, p<0.001), and subsequent multiple comparison usingTukey's HSD test (Tc=14.91 and 18.165) revealed significant increases inthe percentage of apoptotic cells following glutamate treatment (p<0.01,compared to controls). Concurrent treatment of the cultures with GLP-1or exendin-4 significantly protected against glutamate-induced celldeath (both p<0.01, compared to glutamate alone). There were nosignificant differences between the concurrent glutamate/peptidecultures and controls, demonstrating complete protection of neuronsagainst the effects of glutamate.

Animal and Surgical Procedures

Thirty-five adult male Fischer-344 rats weighing approximately 300 geach were housed under controlled light/dark and temperature conditionswith food and water available ad libitum. Rats were anaesthetized withketamine (90 mg/kg) and acepromazine (0.91 mg/kg). Stereotaxic surgerywas carried out as described above. Ibotenic acid dissolved in 0.1 Mphosphate-buffered saline (PBS) was infused unilaterally into the leftlateral branch of the forebrain bundle; referred to as the basal nucleusby Paxinos & Watson (1998) see references, at 10 μg/μl(0.5 μl, 2 sites).Prior pilot examination of the efficacy of this particular batch oftoxin demonstrated that this dose produced a 60% loss of cholineacetyltransferase (ChAT)-positive immunoreactivity in the basalforebrain, with a comparable loss of projections to the cortex. A secondseries of animals receiving infusions of vehicle were used as controls.Each infusion was made over 2.5 min and a further 2.5 min were allowedfor diffusion before the cannula was retracted. After two weeks, animalswere reanaesethetized and stereotaxically implanted with anintracerebroventricular cannula into the right lateral ventricle(AP=−0.8 mm, L=+1.4 mm, V=−4.0 mm).

The cannulae were attached via a catheter to an osmotic minipump (ALZAPharmaceuticals, Mountain View, Calif.). Pumps were filled with 2×10⁻⁸ MGLP-1, 2×10⁻⁹ M exendin-4 or vehicle (artificial cerebrospinal fluid).Both peptides were diluted in vehicle. The pumps were set to deliver0.25 μl over 14 days (total of 5.54 ng GLP-1 at 0.8 nM/kg/min and 0.7 ngexendin-4 at 0.08 nM/kg/min). The brain infusion kits were assembled 5-6h prior to implantation, and left in sterile saline at 37° C. Theminipumps were inserted into a subcutaneous pocket between the shoulderblades, the wounds sutured and the animals allowed to recover. Animalsreceiving infusions of GLP-1 or exendin-4, became modestly aphagic andadipsic, as a result of the insulinotropic nature of the peptides, whichresulted in a slight drop in body weight. This was recouped within 3-4days with the administration of twice daily fluids (0.9% saline) andsoft diet, and by the time of sacrifice there were no differences inbody weight between the groups. On expiry of the minipumps (14 days),animals were terminally anaesthetized 0.1 mg/kg sodium pentobarbitoneand transcardially perfused with 100-150 ml PBS (pH 7.4) followed by250-350 ml 4% paraformaldehyde solution in PBS, at a constant pressureof 100 mm Hg over a period of 15-20 min. The brains were taken forimmunocytochemical assessment and quantification of the lesion-induceddamage and any resulting effects of peptide infusion on the cholinergiccontingent of the basal forebrain.

Immunohistochemistry

Adjacent coronal brain sections were taken at 40 μm thickness, throughthe lesion area, and processed free-floating for ChAT; using thepolyclonal goat anti-ChAT antibody at 1:100 dilution (Chemicon,International Inc., Temecula, Calif.), and glial fibrillary acidicprotein (GFAP); using the polyclonal rabbit anti-GFAP antibody at

1:750 dilution (Chemicon). Visualization of positive immunoreactivitywas carried out using an avidin-biotin/horse radish peroxidase protocol.In addition, one series of sections were stained foracetylcholinesterase (AChE) activity, as a histochemical marker forcholinergic neurons of the basal forebrain using a modified method byGeula and Mesulam, 1989. An additional series of sections were mountedonto gelatin-coated slides and stained with cresyl violet to visualizecell bodies.

ChAT-positive immunoreactive cell bodies in the forebrain area werevisualized under ×100 magnification and manually counted on both sides.The raw counts were corrected with the Abercrombie (1946) formula for anestimate of the total number of cell bodies in the area.Characterization of the cell loss in the basal nucleus as a result ofthe ibotenic acid lesion, was made by comparison of left (lesion side)relative to right (infusion side) counts, and the data presented as thepercent change. The animal names were coded such that cell counts wereformally conducted blind to the experimental condition. Differences weresubjected to analysis of variance, and a posteriori comparisons usingTukey's HSD test as above. Significance was accepted at p<0.05 for allstatistical analyses.

Ibotenic Acid Induced Cholinergic Marker Deficit

Choline acetyltransferase immunoreactivity was used as a marker forcholinergic neurons throughout the basal forebrain. The ChAT antibodystained numerous large multipolar neurons, with a similar size anddistribution to the acetylcholinesterase (AChE) positive cells. Theimmunocytochemical staining had low background and provided a clearpicture of the cell morphology (FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D,FIG. 17E, and FIG. 17F). Injection of ibotenic acid with subsequentinfusion of vehicle resulted in a substantial (43%) loss ofChAT-immunoreactive neurons (FIG. 18, bar 4) over an approximately 1 mmradius from the injection site in the left basal nucleus. Thesham-operated control group receiving vehicle infusion, showed anincrease in the percentage of ChAT-positive cell bodies in the leftbasal nucleus relative to the right basal nucleus (FIG. 18, bar 1).Ibotenate lesioned animals that received GLP-1 or exendin-4 infusionsresulted in a decreased loss of ChAT-immunoreactive cell bodies in theleft basal nucleus relative to those lesioned animals that receivedvehicle infusion. More specifically, infusion of exendin-4 produced adecrease in the loss of ChAT-immunoreactive cell bodies in the leftbasal nucleus, from 43% as was apparent following vehicle infusion, tojust 24% below that of the right basal nucleus (FIG. 18, bar 5).Furthermore, GLP-1 infusion resulted in more striking reversal effects,decreasing the loss of ChAT-positive immunoreactive cell bodies in theleft basal nucleus to just 6% below that of the right basal nucleus(FIG. 18, bar 6). Standard ANOVA demonstrated an overall significanteffect of treatment condition (F=21.363, df=5.28, p<0.001). Multiplecomparisons of peptide vs. vehicle treatment (Tc=14.14 and 19.71)revealed significant improvements in ChAT-immunoreactivity in the leftbasal nucleus following infusion of exendin-4 (p<0.05) and GLP-1(p<0.01) after an ibotenic acid lesion. Although infusion of GLP-1following an ibotenic acid lesion decreased the ChAT-positive cell lossin the left basal nucleus to produce near equal values with the rightside, the overall percent difference was still significantly lower thanthe sham vehicle group (p<0.001). This is likely due to the perceptibleincrease in ChAT-immunoreactivity in the sham group receiving vehicleinfusion (FIG. 18, bar 1).

Separating the left and right ChAT-positive immunoreactive neuronalcounts for the sham vehicle group (Table 6) revealed a significantdifference between left (586±32) and right (478±40) basal nucleiChAT-positive cell counts (p<0.01). Pressure effects from cannulaimplantation and treatment delivery may account for the apparentdecrease in ChAT-immunoreactivity in the right basal nucleus. Theseobservations suggest that any disturbance of tissue integrity, howevermild or non-specific, can produce a functional disruption of ChATimmunoreactivity. Furthermore, such effects may account for the lowerthan anticipated percent loss in ChAT-positive immunoreactivity in theibotenic acid group receiving vehicle infusion (FIG. 18, bar 4). Toexamine this further, rather than comparing ‘within’ groups (i.e., leftvs. right), ‘between’ groups comparisons were performed of left basalnucleus counts (F=6.136, df=5.28, p<0.001; Table 6) for the sham aCSFand ibotenic acid aCSF groups (586±32 and 260±28, respectively). Thebetween group comparisons revealed a 56% loss in ChAT-immunoreactivity.These data indicate that non-specific damage in the right basal nucleusproduced decreases in the number of ChAT-immunoreactive cell bodies,affecting the overall percent loss when comparisons were made withinindividual experimental groups. In addition, there was no significantdifference between groups 10when right basal nucleus ChAT-positive cellcounts were analysed separately (F=0.512, df=5.28, p>0.05, Table 6),implying that such disruption of tissue integrity affected allexperimental groups equally.

TABLE 6 Abercrombie corrected ChAT-positive cell counts in the basalnucleus. Left basal nucleus Right basal nucleus (lesion side) (infusionside) Group [F = 6.136, df = 5,28, [F = 0.512, df = 5,28, (Number ofanimals) p < 0.001] p > 0.05] Sham aCSF (n = 4) 586 ± 32 478 ± 40** Shamexendin-4 (n = 5) 417 ± 49 416 ± 52 Sham GLP-1 (n = 6) 517 ± 50 499 ± 41Lesion aCSF (n = 6) 260 ± 28 461 ± 54*** Lesion exendin-4 (n = 7) 357 ±35 468 ± 30*** Lesion GLP-1 (n = 6) 404 ± 59 423 ± 45

Example 13 Glucagon-Like Peptide-1 Decreases Amyloid-β Peptide (Aβ)Production and Protects Neurons Against Death Induced by Aβ and Iron

The protective effects of GLP-1 and/or exendin-4 following cell deathinduced by Aβ1-42 or Fe2+, and the effects of GLP-1, exendin-4 andexendin-4 WOT on the processing of βAPP between secreted andintracellular forms in vitro, and ultimately on levels of the Aβ peptidein vivo in control mice were tested.

Culture Conditions

PC12 cells were cultured in RPMI 1640 supplemented with 10%heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum(FBS), and 25 mM Hepes buffer as described elsewhere (Lahiri et. al.,2000). All culture media and sera were obtained from MediaTech Inc.(Herndon, Va.). Cells were seeded at approximately 2.0×10⁶ cells/60-mmdish, on cultureware coated in rat-tail collagen (Roche MolecularBiochemicals, Indianapolis, Ind.). Treatments in triplicate began 24 hafter seeding, once cells were well attached. The medium was aspirated,and 3 ml of fresh low serum media containing 0.5% FBS with theappropriate compound(s) was added. Cells were treated with GLP-1 (3.3,33, and 3301 μg/ml) (Bachem, Torrence, Calif.), and two GLP-1 analogues,exendin-4 (0.1, 1.0 and 10 μg/ml) and exendin4-WOT (0.1 and 1.0 μg/ml).Treatment of PC12 cells with NGF (5, 10, 25 and 50 ng/ml) (Promega,Madison, Wis.) was used as a positive control. In addition, 5 ng/ml NGFand 0.1 μg/ml exendin-4 were added simultaneously in combination.Exendin-4 and its analogue exendin 4-WOT were synthesized and assessedto be >95% pure by high-performance liquid chromatography analysis.

Toxicity Assay

Treatment induced cellular toxicity was examined by assay of secretedlactate dehydrogenase (LDH) levels in the conditioned media from bothtreated and untreated PC12 cells (using the LDH kit from Sigma, St.Louis, Mo.). As expected, LDH levels were elevated relative to the mediastandards taken at the start of drug treatment (FIG. 19A). Analysis ofvariance demonstrated an overall significant effect of treatment on LDHsecretion (F=2.22, df=11.35, p<0.001). Subsequent multiple a posterioricomparisons with controls using Tukey's HSD test (Tc=0.42 and 0.51)revealed a single significant increase in LDH secretion following 10μg/mlEx4 treatment (1.65-fold elevation; p<0.01). The possibility oftoxicity as a result of treatment with GLP-1 and analogues, at the dosesand time points used; with the exception of 10 μg/ml exendin-4, can beruled out. This was further substantiated by the cell counts (afterstaining with trypan blue) carried out before and after GLP-1 treatment,which did not demonstrate any significant change in total cell number.

Western Analysis

Following treatment for three days, conditioned media and cell lysates(prepared as described above) from untreated (low serum medium alone)and treated PC12 cells were subjected to Western immunoblot analysisusing the monoclonal antibody, 22C11 (Roche Molecular Biochemicals,Indianapolis, Ind.). The antibody, raised against E. Coli-made βAPPwhose epitope region has been assigned to residues 66-81 in theectoplasmic cysteine-containing domain, recognizes all mature forms ofβAPP found in cell membranes, as well as carboxyl-truncated solubleforms secreted into conditioned media and βAPP-like proteins (APLP).Visualization of the immunoreactive product was carried out bychemiluminescence, hence molecular weight markers were not visible onthe photographic film. Molecular weight identification of theluminescent product was achieved by superimposing the standard molecularweight markers visible on the PVDF membrane. Densitometricquantification of the protein bands was performed using NIH image.

Western immunoblots of cell lysates from treated or untreated cells,revealed multiple higher molecular weight protein bands (100-140 kDa)that likely represent different isoforms of mature βAPP((βAPP695-βAPP770) and/or their post-translationally modifiedderivatives. Secreted APP was detected in conditioned media from treatedand untreated cells, as a 110-120 kDa protein band, likely representingderivatives of βAPP generated by either α- or β-secretase. Thedifferences observed in the profile of immunoreactive bands in theimmunoblots of intracellular proteins, was due neither to the unequalloading of proteins into the gel nor to the uneven transfer of proteinsonto the membrane, as demonstrated by equal β-actin immunoreactivestaining (using the polyclonal β-actin antibody raised against aspecific region at the carboxyl terminus of human β-actin; Santa CruzBiotechnology, Santa Cruz, Calif.) on the same blots. A visual reactionproduct was produced directly on the PVDF membrane using a biotinylatedsecondary antibody. The standard molecular weight marker was thereforevisible, and confirmed β-actin as a single 42 kDa protein band.Equivalent amounts of total proteins were loaded in each lane of the geland the efficiency of the electrophoretic transfer was monitored bystaining the membranes with 0.1% Ponceau S in 5% acetic acid.

Densitometric quantification of the βAPP and sAPP blots are shown inFIG. 19B and FIG. 19C, respectively. One-way analysis of variance of thequantified αAPP levels (top two high molecular weight bands)demonstrated overall significant effects of treatment (F=2.24; df=11.34;p<0.001). Multiple comparisons with controls were conducted usingTukey's HSD test (Tc=47.0 and 55.75) and revealed increases inintracellular levels of βAPP following treatment with both doses of NGF;5 ng/ml (FIG. 19B, bar 1; p<0.01) and 10 ng/ml (FIG. 19B, bar 2;p<0.01). In contrast to NGF, GLP-1 and analogues significantly decreasedintracellular levels of βAPP (FIG. 19B; 10 ng/ml Ex4, bar 5 (p<0.01),1.0 μg/ml Ex4-WOT, bar 10 (p<0.01), 3.3 μg/ml GLP-1, bar 11 (p<0.01), 33μg/ml GLP-1, bar 12 (p<0.05) and 330 μg/ml GLP-1, bar 13 (p<0.01). Thecombination treatment of 5 ng/ml NGF and 0.1 μg/mlEx4 significantlyincreased intracellular βAPP levels relative to untreated cells (FIG.19B, bar 6; p<0.01).

Analysis of the quantified secreted soluble derivatives of βAPP proteindetected in the conditioned medium, revealed overall significant effectsof treatment (F=2.22, df=11.35, p<0.001). Subsequent multiplecomparisons (Tc=20.11 and 23.91) revealed both doses of NGF treatmentresulted in a significant increase in sAPP (5 ng/ml and 10 ng/ml (bothp<0.01); FIG. 19C, bars 1-2). Following a similar pattern tointracellular βAPP levels, all doses of GLP-1 (3.3 μg/ml (p>0.05), 33μg/ml (p<0.01) and 330 μg/ml (p<0.01); FIG. 19C, bars 9-11), Ex4 (0.1μg/ml, 1.0 μg/ml and 10 μg/ml (all p<0.01); FIG. 19C, bars 3-5) andEx4-WOT (0.1 μg/ml and 1.0 μg/ml (both p<0.01); FIG. 19C, bars 7-8)produced decreases in detectable levels of sAPP in conditioned media.The combination NGF and Ex4 treatment (FIG. 19C, bar 6) failed toproduce any significant change in sAPP levels from that of the controlcells.

To investigate whether the decline in βAPP levels following GLP-1treatment could be extrapolated to a decline in secreted Aβ1-40 levels,conditioned media samples were assayed for AβI-40. We have previouslydemonstrated very low basal levels of Aβ1-42 secretion in PC 12 cells,and generally any detectable treatment induced effects do not reachsignificance. In addition, the predominant Aβ peptide secreted is Aβ1-40and in similar studies using neuroblastoma cells (Lahiri et. al., 1998)Aβ1-40 levels were reflective of effects on Aβ1-42, making itunnecessary to look for Aβ1-42 specific changes. Quantified Aβ1-40levels were subjected to one-way analysis of variance, whichdemonstrated significant overall effects of treatment (F=2.22, df=11.35,p<0.001). Multiple comparisons using Tukey's HSD test (Tc=69.91 and83.13) revealed significant increases in Aβ1-40 levels followingtreatment with NGF alone (both p<0.01) or in combination with exendin-4(p<0.01). Treatment with GLP-1, exendin-4 or exendin-4 WOT alone did notsignificantly alter levels of Aβ1-40 production from that of the controlcells.

Whole brain homogenates were assayed for Aβ1-40 levels followingintracerebroventricular infusions of GLP-1, exendin-4, NGF or vehicle innormal control mice. Animals were housed under controlled light/dark andtemperature conditions with food and water available ad libitum. Leandb+/db+ control male mice (n=24) were anaesthetized with 50 mg/kgpentobarbitone and placed in a stereotaxic surgical frame with mouseadaptor (David Kopf Instruments, Tujunga, Calif.) using temporal bonecup holders. Bilateral infusions of GLP-1 (3.3 μg; n=3 and 6.6 μg; n=4),exendin-4 (0.2 μg; n=3), NGF (2 μg; n=5) or vehicle (artificialcerebrospinal fluid; n=9) were made into the lateral ventricles (AP=−0.2mm, L=±1.0 mm, V=−2.5 mm), at 0.25 μl/min over 4 minutes. An additional4 minutes was allowed for diffusion before the cannula was retracted andthe animal sutured and allowed to recover. After 48 h all animals weresacrificed by cervical dislocation, the brains removed and rapidlyfrozen in liquid nitrogen. Brains were pulverized and stored at −80° C.prior to assaying for Aβ levels.

Aβ Assay

Equivalent volumes of conditioned media and whole brain homogenate wereassayed for Aβ1-40 using a sandwich ELISA (Suzuki et. al., 1994). Themonoclonal antibody BAN50 (raised against Aβ1-16) was used as thecapture antibody for all species of Aβ (Aβ1-40 and Aβ1-42), and themonoclonal antibody BA27 was used to specifically detect Aβ1-40 levels.Levels of Aβ1-40 were expressed in pM concentrations for conditionedmedia samples and finol/g for the mouse brain homogenates, as deducedfrom the appropriate standard curve run in parallel with the assay.

All treatments reduced levels of Aβ1-40 compared to vehicle (FIG. 20).Multiple comparisons following significant main effects of treatment(F=10.577, df=4.19, p<0.001) demonstrated Aβ1-40 levels were reducedsignificantly following 6.6 μg GLP-1 (36%, p<0.01) and 2 μg NGF (40%,p<0.01) treatment. All other comparisons failed to reach significance;3.3 μg GLP-1 (16%), 0.2 μg exendin-4 (23%) (Tc=139.00 and 172.34, usinga harmonic mean correction for unequal group sizes)

Primary Hippocampal Cell Culture

Hippocampi were removed from embryonic day 18 Sprague-Dawley rats, andcells were dissociated by mild trypsination and trituration and seededonto polyethyleneimine-coated plastic 35 mm diameter dishes at a densityof approximately 150 cells/mm² of culture surface. Cultures-weremaintained in Neurobasal medium containing B-27 supplements (Gibco BRL,Carlsbad, Calif.), 2 mM L-glutamine, 1 mM Hepes and 0.001% gentamicinsulfate (Sigma, St. Louis, Mo.) in a 6% CO₂/94% room air atmosphere at37° C. When maintained under these conditions, the hippocampal culturesconsisted of approximately 95% neurons and 5% astrocytes as determinedby immunostaining with antibodies against the neuronal antigen NeuN andthe astrocyte protein glial fibrillary acidic protein. Aβ1-42 waspurchased from Bachem (Torrance, Calif.) and was prepared as a 1 mMstock solution in sterile water. FeSO4 was prepared as a 200 μM stock insterile water. GLP-1 and exendin-4 were prepared as 500× stocks insaline.

Neuronal survival was quantified as described previously (Mark et. al.,1997). Briefly, viable neurons in premarked fields (10× objective) werecounted before experimental treatment and at specified time pointsthereafter. Hippocampal neurons were pretreated with GLP-1 (0, 1, 5, 10and 20 nM) or exendin-4 (0, 50, 100, 200 and 500 nM) for 2 hours.Cultures were then exposed to 2 μM Aβ (1-42) or 1 μM Fe²⁺ for 24 hours.Neurons with intact neurites of uniform diameter and a cell body with asmooth round appearance were considered viable, whereas neurons withfragmented neurites and vacuolated soma were considered non-viable. Thecounting was done without knowledge of culture treatment history.

Exposure of cultured hippocampal cells to Aβ1-42 or to iron (whichinduces hydroxyl radical production and membrane lipid peroxidation)resulted in the death of 55-75% of the neurons during a 24 h timeperiod. To determine if GLP-1 or exendin-4 could protect neurons againstcell death induced by Aβ1-42 and/or Fe²⁺, cells were pretreated withincreasing concentrations of GLP-1 and exendin-4, and then exposed toAβ1-42 or Fe²⁺. GLP-1 protected neurons against death induced by Aβ1-42and Fe²⁺ with a maximum effect occurring with 10 nM GLP-1. Exendin-4also protected neurons against death induced by Aβ1-42 and Fe²⁺, but wasless potent, with a maximum effect occurring with 200 nM exendin-4.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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What is claimed is: 1.-87. (canceled)
 88. A method of treating a subjectwith a neurodegenerative disease or of reducing one or more symptoms ofa neurodegenerative disease in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of apolypeptide comprising GLP-1, exendin-4, or a therapeutically effectiveGLP-1 or exendin-4 analogue, wherein the polypeptide binds to andactivates a receptor that binds GLP-1, exendin-4, or both, and whereinthe neurodegenerative disease is multiple sclerosis. 89.-90. (canceled)91. The method of claim 88, wherein the polypeptide is insulinotropic.92. The method of claim 88, wherein the polypeptide is longer actingthan GLP-1.
 93. The method of claim 88, wherein the polypeptide has agreater binding affinity for the GLP-1 receptor than does GLP-1.
 94. Themethod of claim 88, wherein the polypeptide is selected from the groupconsisting of SEQ ID NOs: 9, 42-48, and 50-52.
 95. The method of claim88, wherein the polypeptide comprises GLP-1 or a therapeuticallyeffective GLP-1 analogue.
 96. The method of claim 95, wherein thepolypeptide is selected from the group consisting of SEQ ID NOs: 5-6 and8.
 97. The method of claim 95, wherein the polypeptide isinsulinotropic.
 98. The method of claim 95, wherein the polypeptide islonger acting than GLP-1.
 99. The method of claim 95, wherein thepolypeptide has a greater binding affinity for the GLP-1 receptor thandoes GLP-1.
 100. The method of claim 88, wherein the polypeptidecomprises exendin-4 or a therapeutically effective exendin-4 analogue.101. The method of claim 100, wherein the polypeptide is selected fromthe group consisting of SEQ ID NOs: 10-12 and
 33. 102. The method ofclaim 100, wherein the polypeptide is insulinotropic.
 103. The method ofclaim 100, wherein the polypeptide is longer acting than GLP-1.
 104. Themethod of claim 101, wherein the polypeptide has a greater bindingaffinity for the GLP-1 receptor than does GLP-1.
 105. A method fortreating a neurodegenerative disease in a human in need thereof,comprising administering a therapeutically effective amount of exendin-4to the human to treat the neurodegenerative disease, wherein theneurodegenerative disease is multiple sclerosis.